Science.gov

Sample records for group proteins antagonistically

  1. Toll-like receptor 4 (TLR4) antagonist eritoran tetrasodium attenuates liver ischemia and reperfusion injury through inhibition of high-mobility group box protein B1 (HMGB1) signaling.

    PubMed

    Mcdonald, Kerry-Ann; Huang, Hai; Tohme, Samer; Loughran, Patricia; Ferrero, Kimberly; Billiar, Timothy; Tsung, Allan

    2014-01-01

    Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1-TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1's release from hypoxic hepatocytes in vitro and thereby weakened HMGB1's activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury. PMID:25375408

  2. Toll-like Receptor 4 (TLR4) Antagonist Eritoran Tetrasodium Attenuates Liver Ischemia and Reperfusion Injury through Inhibition of High-Mobility Group Box Protein B1 (HMGB1) Signaling

    PubMed Central

    McDonald, Kerry-Ann; Huang, Hai; Tohme, Samer; Loughran, Patricia; Ferrero, Kimberly; Billiar, Timothy; Tsung, Allan

    2014-01-01

    Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1–TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1’s release from hypoxic hepatocytes in vitro and thereby weakened HMGB1’s activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury. PMID:25375408

  3. Small molecular weight protein-protein interaction antagonists: an insurmountable challenge?

    PubMed

    Dömling, Alexander

    2008-06-01

    Several years ago small molecular weight protein-protein interaction (PPI) antagonists were considered as the Mount Everest in drug discovery and generally regarded as too difficult to be targeted. However, recent industrial and academic research has produced a great number of new antagonists of diverse PPIs. This review structurally analyses small molecular weight PPI antagonists and their particular targets as well as tools to discover such compounds. Besides general discussions there will be a focus on the PPI p53/mdm2. PMID:18501203

  4. Proneurogenic Group II mGluR antagonist improves learning and reduces anxiety in Alzheimer Aβ oligomer mouse.

    PubMed

    Kim, S H; Steele, J W; Lee, S W; Clemenson, G D; Carter, T A; Treuner, K; Gadient, R; Wedel, P; Glabe, C; Barlow, C; Ehrlich, M E; Gage, F H; Gandy, S

    2014-11-01

    Proneurogenic compounds have recently shown promise in some mouse models of Alzheimer's pathology. Antagonists at Group II metabotropic glutamate receptors (Group II mGluR: mGlu2, mGlu3) are reported to stimulate neurogenesis. Agonists at those receptors trigger γ-secretase-inhibitor-sensitive biogenesis of Aβ42 peptides from isolated synaptic terminals, which is selectively suppressed by antagonist pretreatment. We have assessed the therapeutic potential of chronic pharmacological inhibition of Group II mGluR in Dutch APP (Alzheimer's amyloid precursor protein E693Q) transgenic mice that accumulate Dutch amyloid-β (Aβ) oligomers but never develop Aβ plaques. BCI-838 is a clinically well-tolerated, orally bioavailable, investigational prodrug that delivers to the brain BCI-632, the active Group II mGluR antagonist metabolite. Dutch Aβ-oligomer-forming APP transgenic mice (APP E693Q) were dosed with BCI-838 for 3 months. Chronic treatment with BCI-838 was associated with reversal of transgene-related amnestic behavior, reduction in anxiety, reduction in levels of brain Aβ monomers and oligomers, and stimulation of hippocampal neurogenesis. Group II mGluR inhibition may offer a unique package of relevant properties as an Alzheimer's disease therapeutic or prophylactic by providing both attenuation of neuropathology and stimulation of repair. PMID:25113378

  5. Structure-Based Design of a Periplasmic Binding Protein Antagonist that Prevents Domain Closure

    SciTech Connect

    Borrok, M. Jack; Zhu, Yimin; Forest, Katrina T.; Kiessling, Laura L.

    2009-07-31

    Many receptors undergo ligand-induced conformational changes to initiate signal transduction. Periplasmic binding proteins (PBPs) are bacterial receptors that exhibit dramatic conformational changes upon ligand binding. These proteins mediate a wide variety of fundamental processes including transport, chemotaxis, and quorum sensing. Despite the importance of these receptors, no PBP antagonists have been identified and characterized. In this study, we identify 3-O-methyl-D-glucose as an antagonist of glucose/galactose-binding protein and demonstrate that it inhibits glucose chemotaxis in E. coli. Using small-angle X-ray scattering and X-ray crystallography, we show that this antagonist acts as a wedge. It prevents the large-scale domain closure that gives rise to the active signaling state. Guided by these results and the structures of open and closed glucose/galactose-binding protein, we designed and synthesized an antagonist composed of two linked glucose residues. These findings provide a blueprint for the design of new bacterial PBP inhibitors. Given the key role of PBPs in microbial physiology, we anticipate that PBP antagonists will have widespread uses as probes and antimicrobial agents.

  6. Convulsant and anticonvulsant actions of agonists and antagonists of group III mGluRs.

    PubMed

    Ghauri, M; Chapman, A G; Meldrum, B S

    1996-06-17

    Group III metabotropic glutamate receptors (mGluR4, 6, 7, 8) are negatively coupled to adenylate cyclase and, when activated presynaptically, decrease the release of glutamate and GABA. We have used intracerebroventricular injections of agonists and antagonists believed to act selectively on these receptors to study the pro- or anti-convulsant effects of mGluR III activation in nonepileptic (Swiss-Webster) and epileptic (DBA/2) mice. In both mouse strains the prototypic agonists L-2-amino-4-phosphonobutanoate (LAP4) and L-serine-O-phosphate are proconvulsant. The supposed antagonists (S)-2-methyl-2-amino-4-phosphonobutanoate (MAP4) and (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG), have a predominantly proconvulsant effect. (S)-alpha-methyl-3-carboxyphenylalanine, which is a potent and selective antagonist for LAP4 in the cortex, is anticonvulsant in DBA/2 mice and decreases the convulsant effect of N-methyl-D-aspartate, 3,5-dihydroxyphenylglycine, LAP4 and MPPG in Swiss-Webster mice. These data suggest that reduced inhibitory transmission may be more significant than reduced synaptic release of glutamate following group III mGluR activation. PMID:8856700

  7. Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

    PubMed

    Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

    2016-06-01

    Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. PMID:27167364

  8. All About the Protein Foods Group

    MedlinePlus

    ... Waste Food Safety Newsroom Dietary Guidelines Communicator’s Guide All about the Protein Foods Group You are here Home / MyPlate / Protein Foods All about the Protein Foods Group Print Share What ...

  9. A new approach to assess the spasticity in hamstrings muscles using mechanomyography antagonist muscular group.

    PubMed

    Krueger, Eddy; Scheeren, Eduardo M; Nogueira-Neto, Guilherme N; Button, Vera Lúcia da S N; Nohama, Percy

    2012-01-01

    Several pathologies can cause muscle spasticity. Modified Ashworth scale (MAS) can rank spasticity, however its results depend on the physician subjective evaluation. This study aims to show a new approach to spasticity assessment by means of MMG analysis of hamstrings antagonist muscle group (quadriceps muscle). Four subjects participated in the study, divided into two groups regarding MAS (MAS0 and MAS1). MMG sensors were positioned over the muscle belly of rectus femoris (RF), vastus lateralis (VL) and vastus medialis (VM) muscles. The range of movement was acquired with an electrogoniometer placed laterally to the knee. The system was based on a LabVIEW acquisition program and the MMG sensors were built with triaxial accelerometers. The subjects were submitted to stretching reflexes and the integral of the MMG (MMG(INT)) signal was calculated to analysis. The results showed that the MMG(INT) was greater to MAS1 than to MAS0 [muscle RF (p = 0.004), VL (p = 0.001) and VM (p = 0.007)]. The results showed that MMG was viable to detect a muscular tonus increase in antagonist muscular group (quadriceps femoris) of spinal cord injured volunteers. PMID:23366325

  10. RNA aptamers as effective protein antagonists in a multicellular organism

    PubMed Central

    Shi, Hua; Hoffman, Bryan E.; Lis, John T.

    1999-01-01

    RNA aptamers selected against proteins can be used to modulate specific protein function. Expression of such reagents in cells and whole organisms could provide a means of dissecting and controlling molecular mechanisms in vivo. We demonstrate that Drosophila B52 protein can be specifically inhibited in vitro and in vivo by a multivalent RNA aptamer. This inhibitory aptamer RNA binds B52 avidly and inhibits B52-stimulated pre-mRNA splicing. It can be expressed in cultured cells and whole animals in a stable form that accumulates up to 10% of total mRNA. It binds B52 in vivo and suppresses all phenotypes caused by B52 overexpression. The strategies presented here should prove general in design and expression of functional and therapeutic RNAs. PMID:10468557

  11. Oxytocin differentially modulates compromise and competitive approach but not withdrawal to antagonists from own vs. rivaling other groups.

    PubMed

    Ten Velden, Femke S; Baas, Matthijs; Shalvi, Shaul; Kret, Mariska E; De Dreu, Carsten K W

    2014-09-11

    In humans, oxytocin promotes cognitive and motivational tendencies that benefit the groups on which humans depend for their survival and prosperity. Here we examined decision making in an incentivized two-player poker game with either an in-group or out-group antagonist. Sixty nine healthy males received 24 IU oxytocin or matching placebo, and played four rounds of a simplified poker game. On each round they received either low or high value cards to create differences in competitive strength, and then responded to a bet placed by their (simulated) (in-group or out-group) antagonist. Under placebo, participants withdrew and competed depending on their own (low vs. high) competitive strength, regardless of their antagonist's group membership. Under oxytocin, however, participants settled more and competed less with an in-group as compared to an out-group antagonist; withdrawal was unaffected by group membership. We conclude that oxytocin sensitizes humans to the group membership of their interaction partner, rendering them relatively more benevolent and less competitive towards those seen as belonging to their own group. This article is part of a Special Issue entitled Oxytocin and Social Behav. PMID:24055737

  12. Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity.

    PubMed

    Kathum, Omer Abid; Schräder, Tobias; Anhlan, Darisuren; Nordhoff, Carolin; Liedmann, Swantje; Pande, Amit; Mellmann, Alexander; Ehrhardt, Christina; Wixler, Viktor; Ludwig, Stephan

    2016-06-01

    Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed. PMID:26687707

  13. Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists

    PubMed Central

    Cheng, Han; Lear-Rooney, Calli M.; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W.; Olinger, Gene G.

    2015-01-01

    ABSTRACT Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. IMPORTANCE Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of

  14. Screening Bicyclic Peptide Libraries for Protein-Protein Interaction Inhibitors: Discovery of a Tumor Necrosis Factor-alpha Antagonist

    PubMed Central

    Rhodes, Curran A.; Liu, Yusen; Pei, Dehua

    2013-01-01

    Protein-protein interactions represent a new class of exciting but challenging drug targets, because their large, flat binding sites lack well defined pockets for small molecules to bind. We report here a methodology for chemical synthesis and screening of large combinatorial libraries of bicyclic peptides displayed on rigid small-molecule scaffolds. With planar trimesic acid as the scaffold, the resulting bicyclic peptides are effective for binding to protein surfaces such as the interfaces of protein-protein interactions. Screening of a bicyclic peptide library against tumor necrosis factor-alpha (TNFα) identified a potent antagonist that inhibits the TNFα-TNFα receptor interaction and protects cells from TNFα-induced cell death. Bicyclic peptides of this type may provide a general solution for inhibition of protein-protein interactions. PMID:23865589

  15. The Rabies Virus Interferon Antagonist P Protein Interacts with Activated STAT3 and Inhibits Gp130 Receptor Signaling

    PubMed Central

    Lieu, Kim G.; Brice, Aaron; Wiltzer, Linda; Hirst, Bevan; Jans, David A.; Blondel, Danielle

    2013-01-01

    Immune evasion by rabies virus depends on targeting of the signal transducers and activator of transcription 1 (STAT1) and STAT2 proteins by the viral interferon antagonist P protein, but targeting of other STAT proteins has not been investigated. Here, we find that P protein associates with activated STAT3 and inhibits STAT3 nuclear accumulation and Gp130-dependent signaling. This is the first report of STAT3 targeting by the interferon antagonist of a virus other than a paramyxovirus, indicating that STAT3 antagonism is important to a range of human-pathogenic viruses. PMID:23698294

  16. Targeting inhibitor of apoptosis proteins in combination with ErbB antagonists in breast cancer

    PubMed Central

    Foster, Fiona M; Owens, Thomas W; Tanianis-Hughes, Jolanta; Clarke, Robert B; Brennan, Keith; Bundred, Nigel J; Streuli, Charles H

    2009-01-01

    Introduction Inhibitor of apoptosis (IAPs) proteins are a family of proteins that can block apoptosis in normal cells and have been suggested to cause resistance to apoptosis in cancer. Overexpression of oncogenic receptor tyrosine kinases is common in breast cancer; in particular 20% of all cases show elevated Her2. Despite clinical success with the use of targeted therapies, such as Trastuzumab, only up to 35% of Her2-positive patients initially respond. We reasoned that IAP-mediated apoptosis resistance might contribute to this insensitivity to receptor tyrosine kinase therapy, in particular ErbB antagonists. Here we examine the levels of IAPs in breast cancer and evaluate whether targeting IAPs can enhance apoptosis in response to growth factor receptor antagonists and TRAIL. Methods IAP levels were examined in a breast cancer cell line panel and in patient samples. IAPs were inhibited using siRNA or cell permeable mimetics of endogenous inhibitors. Cells were then exposed to TRAIL, Trastuzumab, Lapatinib, or Gefitinib for 48 hours. Examining nuclear morphology and staining for cleaved caspase 3 was used to score apoptosis. Proliferation was examined by Ki67 staining. Results Four members of the IAP family, Survivin, XIAP, cIAP1 and cIAP2, were all expressed to varying extents in breast cancer cell lines or tumours. MDAMB468, BT474 and BT20 cells all expressed XIAP to varying extents. Depleting the cells of XIAP overcame the intrinsic resistance of BT20 and MDAMB468 cells to TRAIL. Moreover, siRNA-based depletion of XIAP or use of a Smac mimetic to target multiple IAPs increased apoptosis in response to the ErbB antagonists, Trastuzumab, Lapatinib or Gefitinib in Her2-overexpressing BT474 cells, or Gefitinib in EGFR-overexpressing MDAMB468 cells. Conclusions The novel findings of this study are that multiple IAPs are concomitantly expressed in breast cancers, and that, in combination with clinically relevant Her2 treatments, IAP antagonists promote apoptosis

  17. 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

  18. Purification of human recombinant interleukin 1 receptor antagonist proteins upon Bacillus subtilis sporulation.

    PubMed

    Maurizi, G; Di Cioccio, V; Macchia, G; Bossù, P; Bizzarri, C; Visconti, U; Boraschi, D; Tagliabue, A; Ruggiero, P

    1997-03-01

    Human interleukin 1 receptor antagonist (IL-1ra) and IL-1ra mutants were constitutively expressed in recombinant Bacillus subtilis in endocellular and active form. In order to optimize the purification of the recombinant proteins, a new method has been developed. After bacterial growth in fermenter, release of recombinant protein was achieved by starvation-induced sporulation. The sporulation supernatant was recovered by centrifugation, filtered, and subjected sequentially to cation- and anion-exchange chromatography. Alternatively, the fermenter's contents were directly subjected to expanded bed adsorption on a Streamline cation-exchange column, thus avoiding the centrifugation and filtration steps. Up to 88 mg of biological active purified recombinant protein per liter of culture was obtained, with a 72-79% recovery and 98% purity, depending on the molecule. By using the method described here, it is possible to achieve a spontaneous release of recombinant proteins expressed endocellularly at high levels in B. subtilis without need of a cell breakage step. Thus, this method could allow purification of the endocellular recombinant protein as if it were secreted. Furthermore, when using the expanded bed adsorption, highly purified protein was obtained in only two steps after sporulation. Among the advantages of the method, one of the most relevant is the possibility of keeping the system closed up to completion of the first purification step. PMID:9056487

  19. Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase.

    PubMed Central

    Polya, G M; Chandra, S; Condron, R

    1993-01-01

    A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cation-exchange column. This RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by denaturation in the presence of guanidinium HCl and mercaptoethanol and subsequent reverse-phase HPLC on a C8 column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Ca(2+)-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA preparation, RCA1, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 microM, respectively). N-terminal sequencing by sequential Edman degradation of RCA1, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3' (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent to the sites of proteolytic precursor cleavage. PMID:8278508

  20. Group II Introns and Their Protein Collaborators

    NASA Astrophysics Data System (ADS)

    Solem, Amanda; Zingler, Nora; Pyle, Anna Marie; Li-Pook-Than, Jennifer

    Group II introns are an abundant class of autocatalytic introns that excise themselves from precursor mRNAs. Although group II introns are catalytic RNAs, they require the assistance of proteins for efficient splicing in vivo. Proteins that facilitate splicing of organellar group II introns fall into two main categories: intron-encoded maturases and host-encoded proteins. This chapter will focus on the host proteins that group II introns recruited to ensure their function. It will discuss the great diversity of these proteins, define common features, and describe different strategies employed to achieve specificity. Special emphasis will be placed on DEAD-box ATPases, currently the best studied example of host-encoded proteins with a role in group II intron splicing. Since the exact mechanisms by which splicing is facilitated is not known for any of the host proteins, general mechanistic strategies for protein-mediated RNA folding are described and assessed for their potential role in group II intron splicing.

  1. Calcium antagonists.

    PubMed

    Grossman, Ehud; Messerli, Franz H

    2004-01-01

    Calcium antagonists were introduced for the treatment of hypertension in the 1980s. Their use was subsequently expanded to additional disorders, such as angina pectoris, paroxysmal supraventricular tachycardias, hypertrophic cardiomyopathy, Raynaud phenomenon, pulmonary hypertension, diffuse esophageal spasms, and migraine. Calcium antagonists as a group are heterogeneous and include 3 main classes--phenylalkylamines, benzothiazepines, and dihydropyridines--that differ in their molecular structure, sites and modes of action, and effects on various other cardiovascular functions. Calcium antagonists lower blood pressure mainly through vasodilation and reduction of peripheral resistance. They maintain blood flow to vital organs, and are safe in patients with renal impairment. Unlike diuretics and beta-blockers, calcium antagonists do not impair glucose metabolism or lipid profile and may even attenuate the development of arteriosclerotic lesions. In long-term follow-up, patients treated with calcium antagonists had development of less overt diabetes mellitus than those who were treated with diuretics and beta-blockers. Moreover, calcium antagonists are able to reduce left ventricular mass and are effective in improving anginal pain. Recent prospective randomized studies attested to the beneficial effects of calcium antagonists in hypertensive patients. In comparison with placebo, calcium antagonist-based therapy reduced major cardiovascular events and cardiovascular death significantly in elderly hypertensive patients and in diabetic patients. In several comparative studies in hypertensive patients, treatment with calcium antagonists was equally effective as treatment with diuretics, beta-blockers, or angiotensin-converting enzyme inhibitors. From these studies, it seems that a calcium antagonist-based regimen is superior to other regimens in preventing stroke, equivalent in preventing ischemic heart disease, and inferior in preventing congestive heart failure

  2. Pushing the threshold: How NMDAR antagonists induce homeostasis through protein synthesis to remedy depression.

    PubMed

    Raab-Graham, Kimberly F; Workman, Emily R; Namjoshi, Sanjeev; Niere, Farr

    2016-09-15

    Healthy neurons have an optimal operating range, coded globally by the frequency of action potentials or locally by calcium. The maintenance of this range is governed by homeostatic plasticity. Here, we discuss how new approaches to treat depression alter synaptic activity. These approaches induce the neuron to recruit homeostatic mechanisms to relieve depression. Homeostasis generally implies that the direction of activity necessary to restore the neuron's critical operating range is opposite in direction to its current activity pattern. Unconventional antidepressant therapies-deep brain stimulation and NMDAR antagonists-alter the neuron's "depressed" state by pushing the neuron's current activity in the same direction but to the extreme edge. These therapies rally the intrinsic drive of neurons in the opposite direction, thereby allowing the cell to return to baseline activity, form new synapses, and restore proper communication. In this review, we discuss seminal studies on protein synthesis dependent homeostatic plasticity and their contribution to our understanding of molecular mechanisms underlying the effectiveness of NMDAR antagonists as rapid antidepressants. Rapid antidepressant efficacy is likely to require a cascade of mRNA translational regulation. Emerging evidence suggests that changes in synaptic strength or intrinsic excitability converge on the same protein synthesis pathways, relieving depressive symptoms. Thus, we address the question: Are there multiple homeostatic mechanisms that induce the neuron and neuronal circuits to self-correct to regulate mood in vivo? Targeting alternative ways to induce homeostatic protein synthesis may provide, faster, safer, and longer lasting antidepressants. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease. PMID:27125595

  3. The antagonistic effect of Banana bunchy top virus multifunctional protein B4 against Fusarium oxysporum.

    PubMed

    Zhuang, Jun; Coates, Christopher J; Mao, Qianzhuo; Wu, Zujian; Xie, Lianhui

    2016-06-01

    The viral-induced banana bunchy top disease and the fungal-induced banana blight are two major causes of concern for industrial scale production of bananas. Banana blight is particularly troublesome, affecting ∼80% of crops worldwide. Strict guidelines and protocols are in place in order to ameliorate the effects of this devastating disease, yet little success has been achieved. From the data presented here, we have found that Banana bunchy top virus (BBTV)-infected bananas are more resistant to Fusarium oxysporum f. sp. cubense (Foc). BBTV appears to be antagonistic towards Foc, thus improving the survivability of plants against blight. The BBTV suppressor of RNA silencing, namely protein B4, displays fungicidal properties in vitro. Furthermore, transgenic tomatoes expressing green fluorescent protein (GFP)-tagged protein B4 demonstrate enhanced resistance to F. oxysporum f. sp. lycopersici (Fol). Differential gene expression analysis indicates that increased numbers of photogenesis-related gene transcripts are present in dark-green leaves of B4-GFP-modified tomato plants relative to those found in WT plants. Conversely, the transcript abundance of immunity-related genes is substantially lower in transgenic tomatoes compared with WT plants, suggesting that plant defences may be influenced by protein B4. This viral-fungal interaction provides new insights into microbial community dynamics within a single host and has potential commercial value for the breeding of transgenic resistance to Fusarium-related blight/wilt. PMID:26369403

  4. Mediastinal Yolk Sac Tumor Producing Protein Induced by Vitamin K Absence or Antagonist-II.

    PubMed

    Akutsu, Noriyuki; Adachi, Yasushi; Isosaka, Mai; Mita, Hiroaki; Takagi, Hideyasu; Sasaki, Shigeru; Yamamoto, Hiroyuki; Arimura, Yoshiaki; Ishii, Yoshifumi; Masumori, Naoya; Endo, Takao; Shinomura, Yasuhisa

    2015-01-01

    Extragonadal yolk sac tumors (YSTs) are rare. We herein report the case of a 66-year-old man with mediastinal, lung and liver tumors. The largest mass was located in the liver and contained a high concentration of protein induced by vitamin K absence or antagonist-II (PIVKA-II) and alpha-fetoprotein. Therefore, the lesion was difficult to distinguish from hepatocellular carcinoma. Finally, YST was diagnosed based on the results of a liver biopsy. Although chemotherapy was effective, the patient died of respiratory failure. The autopsy revealed primary mediastinal YST. In the current report, we describe this case of PIVKA-II-producing YST and review previous cases of PIVKA-II-producing tumors other than hepatoma. PMID:26073245

  5. Discovery of peptidylarginine deiminase-4 substrates by protein array: antagonistic citrullination and methylation of human ribosomal protein S2.

    PubMed

    Guo, Qin; Bedford, Mark T; Fast, Walter

    2011-07-01

    Peptidylarginine deiminase (PAD) catalyzes the posttranslational citrullination of selected proteins in a calcium dependent manner. The PAD4 isoform has been implicated in multiple sclerosis, rheumatoid arthritis, some types of cancer, and plays a role in gene regulation. However, the substrate selectivity of PAD4 is not well defined, nor is the impact of citrullination on many other pathways. Here, a high-density protein array is used as a primary screen to identify 40 previously unreported PAD4 substrates, 10 of which are selected and verified in a cell lysate-based secondary assay. One of the most prominent hits, human 40S ribosomal protein S2 (RPS2), is characterized in detail. PAD4 citrullinates the Arg-Gly repeat region of RPS2, which is also an established site for Arg methylation by protein arginine methyltransferase 3 (PRMT3). As in other systems, crosstalk is observed; citrullination and methylation modifications are found to be antagonistic to each other, suggesting a conserved posttranslational regulatory strategy. Both PAD4 and PRMT3 are found to co-sediment with the free 40S ribosomal subunit fraction from cell extracts. These findings are consistent with participation of citrullination in the regulation of RPS2 and ribosome assembly. This application of protein arrays to reveal new PAD4 substrates suggests a role for citrullination in a number of different cellular pathways. PMID:21584310

  6. Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.

    PubMed Central

    Bandara, G; Mueller, G M; Galea-Lauri, J; Tindal, M H; Georgescu, H I; Suchanek, M K; Hung, G L; Glorioso, J C; Robbins, P D; Evans, C H

    1993-01-01

    Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy. Images Fig. 1 Fig. 2 PMID:8248169

  7. Exploiting Free-Energy Minima to Design Novel EphA2 Protein-Protein Antagonists: From Simulation to Experiment and Return.

    PubMed

    Russo, Simonetta; Callegari, Donatella; Incerti, Matteo; Pala, Daniele; Giorgio, Carmine; Brunetti, Jlenia; Bracci, Luisa; Vicini, Paola; Barocelli, Elisabetta; Capoferri, Luigi; Rivara, Silvia; Tognolini, Massimiliano; Mor, Marco; Lodola, Alessio

    2016-06-01

    The free-energy surface (FES) of protein-ligand binding contains information useful for drug design. Here we show how to exploit a free-energy minimum of a protein-ligand complex identified by metadynamics simulations to design a new EphA2 antagonist with improved inhibitory potency. PMID:27139720

  8. Effect of Interdomain Linker Length on an Antagonistic Folding-Unfolding Equilibrium between Two Protein Domains

    PubMed Central

    Cutler, Thomas A.; Mills, Brandon M.; Lubin, David J.; Chong, Lillian T.; Loh, Stewart N.

    2009-01-01

    Fusion of one protein domain with another is a common event in both evolution and protein engineering experiments. When insertion is at an internal site (e.g., a surface loop or turn), as opposed to one of the termini, conformational strain can be introduced into both domains. Strain is manifested by an antagonistic folding-unfolding equilibrium between the two domains, which we previously showed can be parameterized by a coupling free-energy term (ΔGX). The extent of strain is predicted to depend primarily on the ratio of the N-to-C distance of the guest protein to the distance between ends of the surface loop in the host protein. Here, we test that hypothesis by inserting ubiquitin (Ub) into the bacterial ribonuclease barnase (Bn), using peptide linkers from zero to 10 amino acids each. ΔGX values are determined by measuring the extent to which Co2+ binding to an engineered site on the Ub domain destabilizes the Bn domain. All-atom, unforced Langevin dynamics simulations are employed to gain structural insight into the mechanism of mechanically induced unfolding. Experimental and computational results find that the two domains are structurally and energetically uncoupled when linkers are long and that ΔGX increases with decreasing linker length. When the linkers are fewer than two amino acids, strain is so great that one domain unfolds the other. However, the protein is able to refold as dimers and higher-order oligomers. The likely mechanism is a three-dimensional domain swap of the Bn domain, which relieves conformational strain. The simulations suggest that an effective route to mechanical unfolding begins with disruption of the hydrophobic core of Bn near the Ub insertion site. PMID:19038264

  9. Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.

    PubMed

    Stoveken, Hannah M; Bahr, Laura L; Anders, M W; Wojtovich, Andrew P; Smrcka, Alan V; Tall, Gregory G

    2016-09-01

    Adhesion G protein-coupled receptors (aGPCRs) have emerging roles in development and tissue maintenance and is the most prevalent GPCR subclass mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCR extracellular domains contain a conserved subdomain that mediates self-cleavage proximal to the start of the 7-transmembrane domain (7TM). The two receptor protomers, extracellular domain and amino terminal fragment (NTF), and the 7TM or C-terminal fragment remain noncovalently bound at the plasma membrane in a low-activity state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix proteins that dissociate the NTF to reveal the tethered agonist. Given the perceived difficulty in modifying extracellular matrix proteins to create aGPCR probes, we developed a serum response element (SRE)-luciferase-based screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000-compound library comprising known drugs and natural products was screened for GPR56-dependent SRE activation inhibitors that did not inhibit constitutively active Gα13-dependent SRE activation. Dihydromunduletone (DHM), a rotenoid derivative, was validated using cell-free aGPCR/heterotrimeric G protein guanosine 5'-3-O-(thio)triphosphate binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5, which have similar tethered agonists, but not the aGPCR GPR110/ADGRF1, M3 muscarinic acetylcholine, or β2 adrenergic GPCRs. DHM inhibited tethered peptide agonist-stimulated and synthetic peptide agonist-stimulated GPR56 but did not inhibit basal activity, demonstrating that it antagonizes the peptide agonist. DHM is a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic. PMID:27338081

  10. Structure-Guided Discovery of Selective Antagonists for the Chromodomain of Polycomb Repressive Protein CBX7.

    PubMed

    Ren, Chunyan; Smith, Steven G; Yap, Kyoko; Li, SiDe; Li, Jiaojie; Mezei, Mihaly; Rodriguez, Yoel; Vincek, Adam; Aguilo, Francesca; Walsh, Martin J; Zhou, Ming-Ming

    2016-06-01

    The chromobox 7 (CBX7) protein of the polycomb repressive complex 1 (PRC1) functions to repress transcription of tumor suppressor p16 (INK4a) through long noncoding RNA, ANRIL (antisense noncoding RNA in the INK4 locus) directed chromodomain (ChD) binding to trimethylated lysine 27 of histone H3 (H3K27me3), resulting in chromatin compaction at the INK4a/ARF locus. In this study, we report structure-guided discovery of two distinct classes of small-molecule antagonists for the CBX7ChD. Our Class A compounds, a series including analogues of the previously reported MS452, inhibit CBX7ChD/methyl-lysine binding by occupying the H3K27me3 peptide binding site, whereas our Class B compound, the newly discovered MS351, appears to inhibit H3K27me3 binding when CBX7ChD is bound to RNA. Our crystal structure of the CBX7ChD/MS351 complex reveals the molecular details of ligand recognition by the aromatic cage residues that typically engage in methyl-lysine binding. We further demonstrate that MS351 effectively induces transcriptional derepression of CBX7 target genes, including p16 (INK4a) in mouse embryonic stem cells and human prostate cancer PC3 cells. Thus, MS351 represents a new class of ChD antagonists that selectively targets the biologically active form of CBX7 of the PRC1 in long noncoding RNA- and H3K27me3-directed gene transcriptional repression. PMID:27326334

  11. Protein-Protein Interaction Antagonists as Novel Inhibitors of Non-Canonical Polyubiquitylation

    PubMed Central

    Sanclimens, Glòria; Moure, Alejandra; Masip, Isabel; González-Ruiz, Domingo; Rubio, Nuria; Crosas, Bernat; Meca-Cortés, Óscar; Loukili, Noureddine; Plans, Vanessa; Morreale, Antonio; Blanco, Jerónimo; Ortiz, Angel R.; Messeguer, Àngel; Thomson, Timothy M.

    2010-01-01

    Background Several pathways that control cell survival under stress, namely RNF8-dependent DNA damage recognition and repair, PCNA-dependent DNA damage tolerance and activation of NF-κB by extrinsic signals, are regulated by the tagging of key proteins with lysine 63-based polyubiquitylated chains, catalyzed by the conserved ubiquitin conjugating heterodimeric enzyme Ubc13-Uev. Methodology/Principal Findings By applying a selection based on in vivo protein-protein interaction assays of compounds from a combinatorial chemical library followed by virtual screening, we have developed small molecules that efficiently antagonize the Ubc13-Uev1 protein-protein interaction, inhibiting the enzymatic activity of the heterodimer. In mammalian cells, they inhibit lysine 63-type polyubiquitylation of PCNA, inhibit activation of NF-κB by TNF-α and sensitize tumor cells to chemotherapeutic agents. One of these compounds significantly inhibited invasiveness, clonogenicity and tumor growth of prostate cancer cells. Conclusions/Significance This is the first development of pharmacological inhibitors of non-canonical polyubiquitylation that show that these compounds produce selective biological effects with potential therapeutic applications. PMID:20613989

  12. Regulation of alternative splicing of CD45 by antagonistic effects of SR protein splicing factors.

    PubMed

    ten Dam, G B; Zilch, C F; Wallace, D; Wieringa, B; Beverley, P C; Poels, L G; Screaton, G R

    2000-05-15

    CD45 is a transmembrane glycoprotein possessing tyrosine phosphatase activity, which is involved in cell signaling. CD45 is expressed on the surface of most leukocytes and can be alternatively spliced by the inclusion or skipping of three variable exons (4, 5, and 6 or A, B, and C) to produce up to eight isoforms. In T cells, the splicing pattern of CD45 isoforms changes after activation; naive cells express high m.w. isoforms of CD45 which predominantly express exon A (CD45RA), whereas activated cells lose expression of exon A to form low m.w. isoforms of CD45 including CD45RO. Little is known about the specific factors controlling the switch in CD45 splicing which occurs on activation. In this study, we examined the influence of the SR family of splicing factors, which, like CD45, are expressed in tissue-specific patterns and have been shown to modulate the alternative splicing of a variety of transcripts. We show that specific SR proteins have antagonistic effects on CD45 splicing, leading either to exon inclusion or skipping. Furthermore, we were able to demonstrate specific changes in the SR protein expression pattern during T cell activation. PMID:10799890

  13. Fragment-Based Protein-Protein Interaction Antagonists of a Viral Dimeric Protease.

    PubMed

    Gable, Jonathan E; Lee, Gregory M; Acker, Timothy M; Hulce, Kaitlin R; Gonzalez, Eric R; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J; Craik, Charles S

    2016-04-19

    Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. PMID:26822284

  14. Identification of a group of brominated flame retardants as novel androgen receptor antagonists and potential neuronal and endocrine disrupters.

    PubMed

    Kharlyngdoh, Joubert Banjop; Pradhan, Ajay; Asnake, Solomon; Walstad, Anders; Ivarsson, Per; Olsson, Per-Erik

    2015-01-01

    Brominated flame-retardants (BFRs) are used in industrial products to reduce the risk of fire. However, their continuous release into the environment is a concern as they are often persistent, bioaccumulating and toxic. Information on the impact these compounds have on human health and wildlife is limited and only a few of them have been identified to disrupt hormone receptor functions. In the present study we used in silico modeling to determine the interactions of selected BFRs with the human androgen receptor (AR). Three compounds were found to dock into the ligand-binding domain of the human AR and these were further tested using in vitro analysis. Allyl 2,4,6-tribromophenyl ether (ATE), 2-bromoallyl 2,4,6-tribromophenyl ether (BATE) and 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE) were observed to act as AR antagonists. These BFRs have recently been detected in the environment, in house dust and in aquatic animals. The compounds have been detected at high concentrations in both blubber and brain of seals and we therefore also assessed their impact on the expression of L-type amino acid transporter system (LAT) genes, that are needed for amino acid uptake across the blood-brain barrier, as disruption of LAT gene function has been implicated in several brain disorders. The three BFRs down-regulated the expression of AR target genes that encode for prostate specific antigen (PSA), 5α-reductases and β-microseminoprotein. The potency of PSA inhibition was of the same magnitude as the common prostate cancer drugs, demonstrating that these compounds are strong AR antagonists. Western blot analysis of AR protein showed that ATE, BATE and DPTE decreased the 5α-dihydrotestosterone-induced AR protein levels, further confirming that these BFRs act as AR antagonists. The transcription of the LAT genes was altered by the three BFRs, indicating an effect on amino-acid uptake across cellular membranes and blood-brain barrier. This study demonstrated that ATE, BATE

  15. Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography

    SciTech Connect

    Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S.; Kent, Stephen B.H.

    2012-10-23

    Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF{sub 165} to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {l_brace}D-protein antagonist + L-protein form of VEGF-A{r_brace}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 {angstrom}. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 {angstrom}{sup 2} in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

  16. Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography.

    PubMed

    Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S; Kent, Stephen B H

    2012-09-11

    Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2. PMID:22927390

  17. Homo-Oligomerization Facilitates the Interferon-Antagonist Activity of the Ebolavirus VP35 Protein

    PubMed Central

    Reid, St. Patrick; Cárdenas, Washington B.; Basler, Christopher F.

    2014-01-01

    We have identified a putative coiled-coil motif within the amino-terminal half of the ebolavirus VP35 protein. Cross-linking studies demonstrated the ability of VP35 to form trimers, consistent with the presence of a functional coiled-coil motif. VP35 mutants lacking the coiled-coil motif or possessing a mutation designed to disrupt coiled-coil function were defective in oligomerization, as deduced by co-immunoprecipitation studies. VP35 inhibits signaling that activates interferon regulatory factor 3 (IRF-3) and inhibits (IFN)-α/β production. Experiments comparing the ability of VP35 mutants to block IFN responses demonstrated that the VP35 amino-terminus, which retains the putative coiled-coil motif, was unable to inhibit IFN responses, whereas the VP35 carboxy-terminus weakly inhibited the activation of IFN responses. IFN-antagonist function was restored when a heterologous trimerization motif was fused to the carboxy-terminal half of VP35, suggesting that an oligomerization function at the amino-terminus facilitates an “IFNantagonist” function exerted by the carboxy-terminal half of VP35. PMID:16095644

  18. Bluetongue Virus NS4 Protein Is an Interferon Antagonist and a Determinant of Virus Virulence

    PubMed Central

    Ratinier, Maxime; Shaw, Andrew E.; Barry, Gerald; Gu, Quan; Di Gialleonardo, Luigina; Janowicz, Anna; Varela, Mariana; Randall, Richard E.; Caporale, Marco

    2016-01-01

    ABSTRACT Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-β promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence. IMPORTANCE Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein

  19. Discovery of selective probes and antagonists for G-protein-coupled receptors FPR/FPRL1 and GPR30.

    PubMed

    Arterburn, Jeffrey B; Oprea, Tudor I; Prossnitz, Eric R; Edwards, Bruce S; Sklar, Larry A

    2009-01-01

    Recent technological advances in flow cytometry provide a versatile platform for high throughput screening of compound libraries coupled with high-content biological testing and drug discovery. The G protein-coupled receptors (GPCRs) constitute the largest class of signaling molecules in the human genome with frequent roles in disease pathogenesis, yet many examples of orphan receptors with unknown ligands remain. The complex biology and potential for drug discovery within this class provide strong incentives for chemical biology approaches seeking to develop small molecule probes to facilitate elucidation of mechanistic pathways and enable specific manipulation of the activity of individual receptors. We have initiated small molecule probe development projects targeting two distinct families of GPCRs: the formylpeptide receptors (FPR/FPRL1) and G protein-coupled estrogen receptor (GPR30). In each case the assay for compound screening involved the development of an appropriate small molecule fluorescent probe, and the flow cytometry platform provided inherently biological rich assays that enhanced the process of identification and optimization of novel antagonists. The contributions of cheminformatics analysis tools, virtual screening, and synthetic chemistry in synergy with the biomolecular screening program have yielded valuable new chemical probes with high binding affinity, selectivity for the targeted receptor, and potent antagonist activity. This review describes the discovery of novel small molecule antagonists of FPR and FPRL1, and GPR30, and the associated characterization process involving secondary assays, cell based and in vivo studies to define the selectivity and activity of the resulting chemical probes. PMID:19807662

  20. Source memory in rats is impaired by an NMDA receptor antagonist but not by PSD95-nNOS protein-protein interaction inhibitors.

    PubMed

    Smith, Alexandra E; Xu, Zhili; Lai, Yvonne Y; Kulkarni, Pushkar M; Thakur, Ganesh A; Hohmann, Andrea G; Crystal, Jonathon D

    2016-05-15

    Limitations of preclinical models of human memory contribute to the pervasive view that rodent models do not adequately predict therapeutic efficacy in producing cognitive impairments or improvements in humans. We used a source-memory model (i.e., a representation of the origin of information) we developed for use in rats to evaluate possible drug-induced impairments of both spatial memory and higher order memory functions in the same task. Memory impairment represents a major barrier to use of NMDAR antagonists as pharmacotherapies. The scaffolding protein postsynaptic density 95kDa (PSD95) links NMDARs to the neuronal enzyme nitric oxide synthase (nNOS), which catalyzes production of the signaling molecule nitric oxide (NO). Therefore, interrupting PSD95-nNOS protein-protein interactions downstream of NMDARs represents a novel therapeutic strategy to interrupt NMDAR-dependent NO signaling while bypassing unwanted side effects of NMDAR antagonists. We hypothesized that the NMDAR antagonist MK-801 would impair source memory. We also hypothesized that PSD95-nNOS inhibitors (IC87201 and ZL006) would lack the profile of cognitive impairment associated with global NMDAR antagonists. IC87201 and ZL006 suppressed NMDA-stimulated formation of cGMP, a marker of NO production, in cultured hippocampal neurons. MK-801, at doses that did not impair motor function, impaired source memory under conditions in which spatial memory was spared. Thus, source memory was more vulnerable than spatial memory to impairment. By contrast, PSD95-nNOS inhibitors, IC87201 and ZL006, administered at doses that are behaviorally effective in rats, spared source memory, spatial memory, and motor function. Thus, PSD95-nNOS inhibitors are likely to exhibit favorable therapeutic ratios compared to NMDAR antagonists. PMID:26909849

  1. A Potent d-Protein Antagonist of VEGF-A is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo.

    PubMed

    Uppalapati, Maruti; Lee, Dong Jun; Mandal, Kalyaneswar; Li, Hongyan; Miranda, Les P; Lowitz, Joshua; Kenney, John; Adams, Jarrett J; Ault-Riché, Dana; Kent, Stephen B H; Sidhu, Sachdev S

    2016-04-15

    Polypeptides composed entirely of d-amino acids and the achiral amino acid glycine (d-proteins) inherently have in vivo properties that are proposed to be near-optimal for a large molecule therapeutic agent. Specifically, d-proteins are resistant to degradation by proteases and are anticipated to be nonimmunogenic. Furthermore, d-proteins are manufactured chemically and can be engineered to have other desirable properties, such as improved stability, affinity, and pharmacokinetics. Thus, a well-designed d-protein therapeutic would likely have significant advantages over l-protein drugs. Toward the goal of developing d-protein therapeutics, we previously generated RFX001.D, a d-protein antagonist of natural vascular endothelial growth factor A (VEGF-A) that inhibited binding to its receptor. However, RFX001.D is unstable at physiological temperatures (Tm = 33 °C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (Tm > 95 °C), high affinity for VEGF-A (Kd = 6 nM), and improved receptor blocking. Comparison of the two enantiomeric forms of RFX037 revealed that the d-protein is more stable in mouse, monkey, and human plasma and has a longer half-life in vivo in mice. Significantly, RFX037.D was nonimmunogenic in mice, whereas the l-enantiomer generated a strong immune response. These results confirm the potential utility of synthetic d-proteins as alternatives to therapeutic antibodies. PMID:26745345

  2. Effect of plasma protein binding on in vivo activity and brain penetration of glycine/NMDA receptor antagonists.

    PubMed

    Rowley, M; Kulagowski, J J; Watt, A P; Rathbone, D; Stevenson, G I; Carling, R W; Baker, R; Marshall, G R; Kemp, J A; Foster, A C; Grimwood, S; Hargreaves, R; Hurley, C; Saywell, K L; Tricklebank, M D; Leeson, P D

    1997-12-01

    A major issue in designing drugs as antagonists at the glycine site of the NMDA receptor has been to achieve good in vivo activity. A series of 4-hydroxyquinolone glycine antagonists was found to be active in the DBA/2 mouse anticonvulsant assay, but improvements in in vitro affinity were not mirrored by corresponding increases in anticonvulsant activity. Here we show that binding of the compounds to plasma protein limits their brain penetration. Relative binding to the major plasma protein, albumin, was measured in two different ways: by a radioligand binding experiment or using an HPLC assay, for a wide structural range of glycine/NMDA site ligands. These measures of plasma protein binding correlate well (r = 0.84), and the HPLC assay has been used extensively to quantify plasma protein binding. For the 4-hydroxyquinolone series, binding to plasma protein correlates (r = 0.92) with log P (octanol/pH 7.4 buffer) over a range of log P values from 0 to 5. The anticonvulsant activity increases with in vitro affinity, but the slope of a plot of pED50 versus pIC50 is low (0.40); taking plasma protein binding into account in this plot increases the slope to 0.60. This shows that binding to albumin in plasma reduces the amount of compound free to diffuse across the blood-brain barrier. Further evidence comes from three other experiments: (a) Direct measurements of brain/blood ratios for three compounds (2, 16, 26) show the ratio decreases with increasing log R. (b) Warfarin, which competes for albumin binding sites dose-dependently, decreased the ED50 of 26 for protection against seizures induced by NMDLA. (c) Direct measurements of brain penetration using an in situ brain perfusion model in rat to measure the amount of drug crossing the blood-brain barrier showed that compounds 2, 26, and 32 penetrate the brain well in the absence of plasma protein, but this is greatly reduced when the drug is delivered in plasma. In the 4-hydroxyquinolones glycine site binding affinity

  3. Phenylglycine derivatives as antagonists of group III metabotropic glutamate receptors expressed on neonatal rat primary afferent terminals

    PubMed Central

    Miller, Jacqueline C; Howson, Patrick A; Conway, Stuart J; Williams, Richard V; Clark, Barry P; Jane, David E

    2003-01-01

    Three novel phenylglycine analogues; (RS)-α-methyl-3-chloro-4-phosphonophenylglycine (UBP1110), (RS)-α-methyl-3-methoxy-4-phosphonophenylglycine (UBP1111) and (RS)-α-methyl-3-methyl-4-phosphonophenylglycine (UBP1112) antagonised the depression of the fast component of the dorsal root-evoked ventral root potential induced by (S)-AP4 with apparent KD values of: 7.4±2.3, 5.4±0.6 and 5.1±0.3 μM (all n=3), respectively. A Schild analysis of the antagonism of (S)-AP4 induced depression of synaptic transmission by UBP1112 revealed a pA2 value of 5.3 and a slope of 0.81±0.26 (n=9). None of the phenylglycines tested were potent antagonists of responses mediated by group II mGlu receptors (apparent KD values >480 μM). UBP1112 when tested at a concentration of 1 mM had little or no activity on (S)-3,5-DHPG-, NMDA-, AMPA- or kainate-induced responses on motoneurones. PMID:12922940

  4. Small molecular probes for G-protein-coupled C5a receptors: conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a.

    PubMed

    Wong, A K; Finch, A M; Pierens, G K; Craik, D J; Taylor, S M; Fairlie, D P

    1998-08-27

    Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by 1H NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH. OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH.OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity

  5. Synergistic activity of BET protein antagonist-based combinations in mantle cell lymphoma cells sensitive or resistant to ibrutinib.

    PubMed

    Sun, Baohua; Shah, Bhavin; Fiskus, Warren; Qi, Jun; Rajapakshe, Kimal; Coarfa, Cristian; Li, Li; Devaraj, Santhana G T; Sharma, Sunil; Zhang, Liang; Wang, Michael L; Saenz, Dyana T; Krieger, Stephanie; Bradner, James E; Bhalla, Kapil N

    2015-09-24

    Mantle cell lymphoma (MCL) cells exhibit increased B-cell receptor and nuclear factor (NF)-κB activities. The bromodomain and extra-terminal (BET) protein bromodomain 4 is essential for the transcriptional activity of NF-κB. Here, we demonstrate that treatment with the BET protein bromodomain antagonist (BA) JQ1 attenuates MYC and cyclin-dependent kinase (CDK)4/6, inhibits the nuclear RelA levels and the expression of NF-κB target genes, including Bruton tyrosine kinase (BTK) in MCL cells. Although lowering the levels of the antiapoptotic B-cell lymphoma (BCL)2 family proteins, BA treatment induces the proapoptotic protein BIM and exerts dose-dependent lethality against cultured and primary MCL cells. Cotreatment with BA and the BTK inhibitor ibrutinib synergistically induces apoptosis of MCL cells. Compared with each agent alone, cotreatment with BA and ibrutinib markedly improved the median survival of mice engrafted with the MCL cells. BA treatment also induced apoptosis of the in vitro isolated, ibrutinib-resistant MCL cells, which overexpress CDK6, BCL2, Bcl-xL, XIAP, and AKT, but lack ibrutinib resistance-conferring BTK mutation. Cotreatment with BA and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT-199 (BCL2 antagonist) synergistically induced apoptosis of the ibrutinib-resistant MCL cells. These findings highlight and support further in vivo evaluation of the efficacy of the BA-based combinations with these agents against MCL, including ibrutinib-resistant MCL. PMID:26254443

  6. SET antagonist enhances the chemosensitivity of non-small cell lung cancer cells by reactivating protein phosphatase 2A

    PubMed Central

    Hung, Man-Hsin; Wang, Cheng-Yi; Chen, Yen-Lin; Chu, Pei-Yi; Hsiao, Yung-Jen; Tai, Wei-Tien; Chao, Ting-Ting; Yu, Hui-Chuan; Shiau, Chung-Wai; Chen, Kuen-Feng

    2016-01-01

    SET is known as a potent PP2A inhibitor, however, its oncogenic role including its tumorigenic potential and involvement in the development of chemoresistance in non-small cell lung cancer (NSCLC) has not yet been fully discussed. In present study, we investigated the oncogenic role of SET by SET-knockdown and showed that SET silencing impaired cell growth rate, colony formation and tumor sphere formation in A549 cells. Notably, silencing SET enhanced the pro-apoptotic effects of paclitaxel, while ectopic expression of SET diminished the sensitivity of NSCLC cells to paclitaxel. Since the SET protein was shown to affect chemosensitivity, we next examined whether combining a novel SET antagonist, EMQA, sensitized NSCLC cells to paclitaxel. Both the in vitro and in vivo experiments suggested that EMQA and paclitaxel combination treatment was synergistic. Importantly, we found that downregulating p-Akt by inhibiting SET-mediated protein phosphatase 2A (PP2A) inactivation determined the pro-apoptotic effects of EMQA and paclitaxel combination treatment. To dissect the critical site for EMQA functioning, we generated several truncated SET proteins. By analysis of the effects of EMQA on the binding affinities of different truncated SET proteins to PP2A-catalytic subunits, we revealed that the 227–277 amino-acid sequence is critical for EMQA-induced SET inhibition. Our findings demonstrate the critical role of SET in NSCLC, particularly in the development of chemoresistance. The synergistic effects of paclitaxel and the SET antagonist shown in current study encourage further validation of the clinical potential of this combination. PMID:26575017

  7. Synergistic activity of BET protein antagonist-based combinations in mantle cell lymphoma cells sensitive or resistant to ibrutinib

    PubMed Central

    Sun, Baohua; Shah, Bhavin; Fiskus, Warren; Qi, Jun; Rajapakshe, Kimal; Coarfa, Cristian; Li, Li; Devaraj, Santhana G. T.; Sharma, Sunil; Zhang, Liang; Wang, Michael L.; Saenz, Dyana T.; Krieger, Stephanie; Bradner, James E.

    2015-01-01

    Mantle cell lymphoma (MCL) cells exhibit increased B-cell receptor and nuclear factor (NF)-κB activities. The bromodomain and extra-terminal (BET) protein bromodomain 4 is essential for the transcriptional activity of NF-κB. Here, we demonstrate that treatment with the BET protein bromodomain antagonist (BA) JQ1 attenuates MYC and cyclin-dependent kinase (CDK)4/6, inhibits the nuclear RelA levels and the expression of NF-κB target genes, including Bruton tyrosine kinase (BTK) in MCL cells. Although lowering the levels of the antiapoptotic B-cell lymphoma (BCL)2 family proteins, BA treatment induces the proapoptotic protein BIM and exerts dose-dependent lethality against cultured and primary MCL cells. Cotreatment with BA and the BTK inhibitor ibrutinib synergistically induces apoptosis of MCL cells. Compared with each agent alone, cotreatment with BA and ibrutinib markedly improved the median survival of mice engrafted with the MCL cells. BA treatment also induced apoptosis of the in vitro isolated, ibrutinib-resistant MCL cells, which overexpress CDK6, BCL2, Bcl-xL, XIAP, and AKT, but lack ibrutinib resistance-conferring BTK mutation. Cotreatment with BA and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT-199 (BCL2 antagonist) synergistically induced apoptosis of the ibrutinib-resistant MCL cells. These findings highlight and support further in vivo evaluation of the efficacy of the BA-based combinations with these agents against MCL, including ibrutinib-resistant MCL. PMID:26254443

  8. Selective Allosteric Antagonists for the G Protein-Coupled Receptor GPRC6A Based on the 2-Phenylindole Privileged Structure Scaffold.

    PubMed

    Johansson, Henrik; Boesgaard, Michael Worch; Nørskov-Lauritsen, Lenea; Larsen, Inna; Kuhne, Sebastiaan; Gloriam, David E; Bräuner-Osborne, Hans; Sejer Pedersen, Daniel

    2015-11-25

    G protein-coupled receptors (GPCRs) represent a biological target class of fundamental importance in drug therapy. The GPRC6A receptor is a newly deorphanized class C GPCR that we recently reported for the first allosteric antagonists based on the 2-arylindole privileged structure scaffold (e.g., 1-3). Herein, we present the first structure-activity relationship study for the 2-arylindole antagonist 3, comprising the design, synthesis, and pharmacological evaluation of a focused library of 3-substituted 2-arylindoles. In a FRET-based inositol monophosphate (IP1) assay we identified compounds 7, 13e, and 34b as antagonists at the GPRC6A receptor in the low micromolar range and show that 7 and 34b display >9-fold selectivity for the GPRC6A receptor over related GPCRs, making 7 and 34b the most potent and selective antagonists for the GPRC6A receptor reported to date. PMID:26516782

  9. Apoptosis and the FLIP and NF-kappa B proteins as pharmacodynamic criteria for biosimilar TNF-alpha antagonists

    PubMed Central

    Urbano, Paulo César Martins; Soccol, Vanete Thomaz; Azevedo, Valderilio Feijó

    2014-01-01

    Various criteria are necessary to assess the efficacy and safety of biological medications in order to grant companies the right to register these medications with the appropriate bodies that regulate their sale. The imminent expiration of the patents on reference biological products which block the cytokine TNF-α (tumor necrosis factor-α) raises the possibility of bringing so-called biosimilars to the market (similar to the biologicals of reference products). This occurrence is inevitable, but criteria to adequately evaluate these medications are now needed. Even among controversy, there is a demand from publications correlating the pro-apoptotic mechanism of the original TNF-α antagonists (etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol) in the treatment of rheumatoid arthritis and other diseases. In this article, the authors discuss the possibility of utilizing the pro-apoptotic effect correlated with the regulation of the anti-apoptotic proteins FLIP and NF-κB as new criteria for analyzing the pharmacodynamics of possible biosimilar TNF-α antagonists which should be submitted to regulatory agencies for evaluation. PMID:25114503

  10. Cysteinyl Leukotriene Receptor 1/2 Antagonists Nonselectively Modulate Organic Anion Transport by Multidrug Resistance Proteins (MRP1-4).

    PubMed

    Csandl, Mark A; Conseil, Gwenaëlle; Cole, Susan P C

    2016-06-01

    Active efflux of both drugs and organic anion metabolites is mediated by the multidrug resistance proteins (MRPs). MRP1 (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), and MRP4 (ABCC4) have partially overlapping substrate specificities and all transport 17β-estradiol 17-(β-d-glucuronide) (E217βG). The cysteinyl leukotriene receptor 1 (CysLT1R) antagonist MK-571 inhibits all four MRP homologs, but little is known about the modulatory effects of newer leukotriene modifiers (LTMs). Here we examined the effects of seven CysLT1R- and CysLT2R-selective LTMs on E217βG uptake into MRP1-4-enriched inside-out membrane vesicles. Their effects on uptake of an additional physiologic solute were also measured for MRP1 [leukotriene C4 (LTC4)] and MRP4 [prostaglandin E2 (PGE2)]. The two CysLT2R-selective LTMs studied were generally more potent inhibitors than CysLT1R-selective LTMs, but neither class of antagonists showed any MRP selectivity. For E217βG uptake, LTM IC50s ranged from 1.2 to 26.9 μM and were most comparable for MRP1 and MRP4. The LTM rank order inhibitory potencies for E217βG versus LTC4 uptake by MRP1, and E217βG versus PGE2 uptake by MRP4, were also similar. Three of four CysLT1R-selective LTMs also stimulated MRP2 (but not MRP3) transport and thus exerted a concentration-dependent biphasic effect on MRP2. The fourth CysLT1R antagonist, LY171883, only stimulated MRP2 (and MRP3) transport but none of the MRPs were stimulated by either CysLT2R-selective LTM. We conclude that, in contrast to their CysLTR selectivity, CysLTR antagonists show no MRP homolog selectivity, and data should be interpreted cautiously if obtained from LTMs in systems in which more than one MRP is present. PMID:27068271

  11. Sulfhydryl group(s) in the ligand binding site of the D-1 dopamine receptor: specific protection by agonist and antagonist

    SciTech Connect

    Sidhu, A.; Kassis, S.; Kebabian, J.; Fishman, P.H.

    1986-10-21

    An iodinated compound, (/sup 125/I)-8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol, has been recently reported to be a specific ligand for the D-1 dopamine receptor. Due to its high affinity and specific activity, this ligand was chosen for the biochemical characterization of the D-1 receptor. Alkylation of particulate fractions of rat caudate nucleus by N-ethylmaleimide (NEM) caused an inactivation of the D-1 receptor, as measured by diminished binding of the radioligand to the receptor. The inactivation of the receptor sites by NEM was rapid and irreversible, resulting in a 70% net loss of binding sites. On the basis of Scatchard analysis of binding to NEM-treated tissue, the loss in binding sites was due to a net decrease in the receptor number with a 2-fold decrease in the affinity of the receptor for the radioligand. Receptor occupancy by either a D-1 specific agonist or antagonist protected the ligand binding sites from NEM-mediated inactivation. NEM treatment of the receptor in the absence or presence of protective compound abolished the agonist high-affinity state of the receptor as well as membrane adenylate cyclase activity. The above-treated striatal membranes were fused with HeLa membranes and assayed for dopamine-stimulated adenylate cyclase activity. When the sources of D-1 receptors were from agonist-protected membranes, the receptors retained the ability to functionally couple to the HeLa adenylate cyclase. These results suggest that the D-1 dopamine receptor contains NEM-sensitive sulfhydryl group(s) either at or near the vicinity of the ligand binding sites, which are critical for both receptor binding and function.

  12. In vitro and in vivo protein phosphorylation in Avena sativa L. coleoptiles: effects of Ca2+, calmodulin antagonists, and auxin

    NASA Technical Reports Server (NTRS)

    Veluthambi, K.; Poovaiah, B. W.

    1986-01-01

    In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.

  13. Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: glass transition and protein conformation.

    PubMed

    Chang, B S; Beauvais, R M; Dong, A; Carpenter, J F

    1996-07-15

    The effects of glass transition of, and protein conformation in, the dried solid on the storage stability of freeze-dried recombinant human interleukin-1 receptor antagonist (rhIL-1ra) were examined. Glass transition is a temperature-dependent phenomenon. Amorphous materials become hard and brittle at temperatures below their characteristic glass transition temperatures (Tg) such that diffusion of molecules along the matrix is not sufficient to cause large-scale structural changes. To ascertain the importance of the glass transition in protein storage stability, we compared 10 different lyophilized rhIL-1ra formulations, with Tgs ranging from 20 to 56 degrees C, during several weeks of storage at temperatures above and below the samples' Tgs. Protein degradation, both deamidation and aggregation, was greatly accelerated at temperatures above Tg, but for some formulations also arose below Tg. Thus, storage of dried proteins below the Tg is necessary but not sufficient to ensure long-term stability. To examine the effects of protein structure in the dried solid, we prepared formulations with various sucrose concentrations, all of which had a Tg = 66 +/- 2.5 degrees C. With infrared spectroscopy, we determined that the protein lyophilized with /=5% sucrose, conformational change was inhibited during lyophilization. When stored at 50 degrees C, degradation of the freeze-dried protein varied inversely with sucrose concentration. These results indicate that structural changes arising during the lyophilization process led to damage during subsequent storage, even if the storage temperature was less than the Tg. Together the results of these studies document that to obtain optimum stability of dried rhIL-1ra it was necessary to inhibit conformational change during lyophilization and to store at temperatures below the Tg of the dried formulation. PMID:8660705

  14. ACTH Antagonists.

    PubMed

    Clark, Adrian John; Forfar, Rachel; Hussain, Mashal; Jerman, Jeff; McIver, Ed; Taylor, Debra; Chan, Li

    2016-01-01

    Adrenocorticotropin (ACTH) acts via a highly selective receptor that is a member of the melanocortin receptor subfamily of type 1 G protein-coupled receptors. The ACTH receptor, also known as the melanocortin 2 receptor (MC2R), is unusual in that it is absolutely dependent on a small accessory protein, melanocortin receptor accessory protein (MRAP) for cell surface expression and function. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and high degree of ligand specificity suggests that antagonism of this receptor could provide a useful therapeutic aid and a potential investigational tool. Clinical situations in which this could be useful include (1) Cushing's disease and ectopic ACTH syndrome - especially while preparing for definitive treatment of a causative tumor, or in refractory cases, or (2) congenital adrenal hyperplasia - as an adjunct to glucocorticoid replacement. A case for antagonism in other clinical situations in which there is ACTH excess can also be made. In this article, we will explore the scientific and clinical case for an ACTH antagonist, and will review the evidence for existing and recently described peptides and modified peptides in this role. PMID:27547198

  15. ACTH Antagonists

    PubMed Central

    Clark, Adrian John; Forfar, Rachel; Hussain, Mashal; Jerman, Jeff; McIver, Ed; Taylor, Debra; Chan, Li

    2016-01-01

    Adrenocorticotropin (ACTH) acts via a highly selective receptor that is a member of the melanocortin receptor subfamily of type 1 G protein-coupled receptors. The ACTH receptor, also known as the melanocortin 2 receptor (MC2R), is unusual in that it is absolutely dependent on a small accessory protein, melanocortin receptor accessory protein (MRAP) for cell surface expression and function. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and high degree of ligand specificity suggests that antagonism of this receptor could provide a useful therapeutic aid and a potential investigational tool. Clinical situations in which this could be useful include (1) Cushing’s disease and ectopic ACTH syndrome – especially while preparing for definitive treatment of a causative tumor, or in refractory cases, or (2) congenital adrenal hyperplasia – as an adjunct to glucocorticoid replacement. A case for antagonism in other clinical situations in which there is ACTH excess can also be made. In this article, we will explore the scientific and clinical case for an ACTH antagonist, and will review the evidence for existing and recently described peptides and modified peptides in this role. PMID:27547198

  16. Efficient protein knockdown of HaloTag-fused proteins using hybrid molecules consisting of IAP antagonist and HaloTag ligand.

    PubMed

    Tomoshige, Shusuke; Hashimoto, Yuichi; Ishikawa, Minoru

    2016-07-15

    We previously reported a protein knockdown system for HaloTag-fused proteins using hybrid small molecules consisting of alkyl chloride, which binds covalently to HaloTag, linked to BE04 (2), a bestatin (3) derivative with an affinity for cellular inhibitor of apoptosis protein 1 (cIAP1, a kind of ubiquitin ligase). This system addressed several limitations of prior protein knockdown technology, and was applied to degrade two HaloTag-fused proteins. However, the degradation activity of these hybrid small molecules was not potent. Therefore, we set out to improve this system. We report here the design, synthesis and biological evaluation of novel hybrid compounds 4a and 4b consisting of alkyl chloride linked to IAP antagonist MV1 (5). Compounds 4a and 4b were confirmed to reduce the levels of HaloTag-fused tumor necrosis factor α (HaloTag-TNFα), HaloTag-fused cell division control protein 42 (HaloTag-Cdc42), and unfused HaloTag protein in living cells more potently than did BE04-linked compound 1b. Analysis of the mode of action revealed that the reduction of HaloTag-TNFα is proteasome-dependent, and is also dependent on the linker structure between MV1 (5) and alkyl chloride. These compounds appear to induce ubiquitination at the HaloTag moiety of HaloTag-fused proteins. Our results indicate that these newly synthesized MV1-type hybrid compounds, 4a and 4b, are efficient tools for protein knockdown for HaloTag-fused proteins. PMID:27236416

  17. Diversity of Interferon Antagonist Activities Mediated by NSP1 Proteins of Different Rotavirus Strains▿

    PubMed Central

    Arnold, Michelle M.; Patton, John T.

    2011-01-01

    Studies involving limited numbers of rotavirus (RV) strains have shown that the viral gene 5 product, NSP1, can antagonize beta interferon (IFN-β) expression by inducing the degradation of IFN-regulatory factors (IRFs) (IRF3, IRF5, and IRF7) or a component of the E3 ubiquitin ligase complex responsible for activating NF-κB (β-transducin repeat-containing protein [β-TrCP]). To gain a broader perspective of NSP1 activities, we examined various RV strains for the ability to inhibit IFN-β expression in human cells. We found that all strains encoding wild-type NSP1 impeded IFN-β expression but not always through IRF3 degradation. To identify other degradation targets involved in suppressing IFN-β expression, we used transient expression vectors to test the abilities of a diverse collection of NSP1 proteins to target IRF3, IRF5, IRF7, and β-TrCP for degradation. The results indicated that human RVs rely predominantly on the NSP1-induced degradation of IRF5 and IRF7 to suppress IFN signaling, whereas NSP1 proteins of animal RVs tended to target IRF3, IRF5, and IRF7, allowing the animal viruses a broader attack on the IFN-β signaling pathway. The results also suggested that the NSP1-induced degradation of β-TrCP is an uncommon mechanism of subverting IFN-β signaling but is one that can be shared with NSP1 proteins that induce IRF degradation. Our analysis reveals that the activities of NSP1 proteins are diverse, with no obvious correlations between degradations of pairs of target proteins. Thus, RVs have evolved functionally distinct approaches for subverting the host antiviral response, a property consistent with the immense sequence variation noted for NSP1 proteins. PMID:21177809

  18. Gambogic acid is an antagonist of anti-apoptotic Bcl-2-family proteins

    PubMed Central

    Zhai, Dayong; Jin, Chaofang; Shiau, Chung-wai; Kitada, Shinichi; Satterthwait, Arnold C; Reed, John C.

    2008-01-01

    The natural product Gambogic acid (GA) has been reported to have cytotoxic activity against tumor cells in culture, and was identified as an active compound in a cell-based high-throughput screening (HTS) assay for activators of caspases, proteases involved in apoptosis. Using the anti-apoptotic Bcl-2-family protein, Bfl-1, as a target for screening of a library of natural products, we identified GA as a competitive inhibitor that displaced BH3 peptides from Bfl-1 in a fluorescent polarization assay (FPA). Analysis of competition for BH3 peptide binding revealed that GA inhibits all 6 human Bcl-2-family proteins to various extents, with Mcl-1 and Bcl-B the most potently inhibited (concentrations required for 50% inhibition [IC50] <1 μM). Competition for BH3 peptide binding was also confirmed using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. GA functionally inhibited the anti-apoptotic Bcl-2-family proteins, as demonstrated by experiments using isolated mitochondria in which recombinant purified Bcl-2-family proteins suppress SMAC release in vitro, showing that GA neutralizes their suppressive effects on mitochondria in a concentration-dependent manner. GA killed tumor cell lines via an apoptotic mechanism, whereas analogs of GA with greatly reduced potency at BH3 peptide displacement showed little or no cytotoxic activity. However, GA retained cytotoxic activity against bax−/− bak−/− cells in which anti-apoptotic Bcl-2-family proteins lack a cytoprotective phenotype, implying that GA also has additional targets that contribute to its cytotoxic mechanism. Altogether, the findings suggest that suppression of anti-apoptotic Bcl-2-family proteins may be among the cytotoxic mechanisms by which GA kills tumor cells. PMID:18566235

  19. Antagonistic regulation of mRNA expression and splicing by CELF and MBNL proteins

    PubMed Central

    Wang, Eric T.; Ward, Amanda J.; Cherone, Jennifer M.; Giudice, Jimena; Wang, Thomas T.; Treacy, Daniel J.; Lambert, Nicole J.; Freese, Peter; Saxena, Tanvi; Cooper, Thomas A.; Burge, Christopher B.

    2015-01-01

    RNA binding proteins of the conserved CUGBP1, Elav-like factor (CELF) family contribute to heart and skeletal muscle development and are implicated in myotonic dystrophy (DM). To understand their genome-wide functions, we analyzed the transcriptome dynamics following induction of CELF1 or CELF2 in adult mouse heart and of CELF1 in muscle by RNA-seq, complemented by crosslinking/immunoprecipitation-sequencing (CLIP-seq) analysis of mouse cells and tissues to distinguish direct from indirect regulatory targets. We identified hundreds of mRNAs bound in their 3′ UTRs by both CELF1 and the developmentally induced MBNL1 protein, a threefold greater overlap in target messages than expected, including messages involved in development and cell differentiation. The extent of 3′ UTR binding by CELF1 and MBNL1 predicted the degree of mRNA repression or stabilization, respectively, following CELF1 induction. However, CELF1's RNA binding specificity in vitro was not detectably altered by coincubation with recombinant MBNL1. These findings support a model in which CELF and MBNL proteins bind independently to mRNAs but functionally compete to specify down-regulation or localization/stabilization, respectively, of hundreds of mRNA targets. Expression of many alternative 3′ UTR isoforms was altered following CELF1 induction, with 3′ UTR binding associated with down-regulation of isoforms and genes. The splicing of hundreds of alternative exons was oppositely regulated by these proteins, confirming an additional layer of regulatory antagonism previously observed in a handful of cases. The regulatory relationships between CELFs and MBNLs in control of both mRNA abundance and splicing appear to have evolved to enhance developmental transitions in major classes of heart and muscle genes. PMID:25883322

  20. Antitarget Interaction, Acute Toxicity and Protein Binding Studies of Quinazolinedione Sulphonamides as GABA1 Antagonists

    PubMed Central

    Ajeet; Verma, Mansi; Rani, Sangeeta; Kumar, A.

    2016-01-01

    Diseases characterized by recurrent seizures are known as epilepsy. One of the most important mechanisms for handling it is GABA1 receptor mediated inhibition. In the same context while studying the treatment of epilepsy we observed significant effects by derivatives of sulfonamides, which prompted us to design novel derivatives by means of in silico resources with antiepileptic effects. Molecular docking approaches are routinely used in modern drug design to help understand drug–receptor interaction. This study has been performed with the help of Chemdraw Ultra 7.0, GUSAR online tool for IC50 and LD50 predictions, AutoDock Vina (Python Prescription 0.8), and PaDEL software. Results revealed that ligand-protein interaction affinity of all 10 designed molecules ranges from -5.7 Kcal/mol to -5.2 Kcal/mol, which is approximately comparable to pre-existing GABA1 inhibitor i.e. phenytoin (CID: 1775, ligand-protein interaction affinity is -6.5 Kcal/mol). PMID:27168681

  1. Synergistically acting agonists and antagonists of G protein-coupled receptors prevent photoreceptor cell degeneration.

    PubMed

    Chen, Yu; Palczewska, Grazyna; Masuho, Ikuo; Gao, Songqi; Jin, Hui; Dong, Zhiqian; Gieser, Linn; Brooks, Matthew J; Kiser, Philip D; Kern, Timothy S; Martemyanov, Kirill A; Swaroop, Anand; Palczewski, Krzysztof

    2016-01-01

    Photoreceptor cell degeneration leads to visual impairment and blindness in several types of retinal disease. However, the discovery of safe and effective therapeutic strategies conferring photoreceptor cell protection remains challenging. Targeting distinct cellular pathways with low doses of different drugs that produce a functionally synergistic effect could provide a strategy for preventing or treating retinal dystrophies. We took a systems pharmacology approach to identify potential combination therapies using a mouse model of light-induced retinal degeneration. We showed that a combination of U.S. Food and Drug Administration-approved drugs that act on different G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) exhibited synergistic activity that protected retinas from light-induced degeneration even when each drug was administered at a low dose. In functional assays, the combined effects of these drugs were stimulation of Gi/o signaling by activating the dopamine receptors D2R and D4R, as well as inhibition of Gs and Gq signaling by antagonizing D1R and the α1A-adrenergic receptor ADRA1A, respectively. Moreover, transcriptome analyses demonstrated that such combined GPCR-targeted treatments preserved patterns of retinal gene expression that were more similar to those of the normal retina than did higher-dose monotherapy. Our study thus supports a systems pharmacology approach to identify treatments for retinopathies, an approach that could extend to other complex disorders. PMID:27460988

  2. Insulin Stimulates S100B Secretion and These Proteins Antagonistically Modulate Brain Glucose Metabolism.

    PubMed

    Wartchow, Krista Minéia; Tramontina, Ana Carolina; de Souza, Daniela F; Biasibetti, Regina; Bobermin, Larissa D; Gonçalves, Carlos-Alberto

    2016-06-01

    Brain metabolism is highly dependent on glucose, which is derived from the blood circulation and metabolized by the astrocytes and other neural cells via several pathways. Glucose uptake in the brain does not involve insulin-dependent glucose transporters; however, this hormone affects the glucose influx to the brain. Changes in cerebrospinal fluid levels of S100B (an astrocyte-derived protein) have been associated with alterations in glucose metabolism; however, there is no evidence whether insulin modulates glucose metabolism and S100B secretion. Herein, we investigated the effect of S100B on glucose metabolism, measuring D-(3)H-glucose incorporation in two preparations, C6 glioma cells and acute hippocampal slices, and we also investigated the effect of insulin on S100B secretion. Our results showed that: (a) S100B at physiological levels decreases glucose uptake, through the multiligand receptor RAGE and mitogen-activated protein kinase/ERK signaling, and (b) insulin stimulated S100B secretion via PI3K signaling. Our findings indicate the existence of insulin-S100B modulation of glucose utilization in the brain tissue, and may improve our understanding of glucose metabolism in several conditions such as ketosis, streptozotocin-induced dementia and pharmacological exposure to antipsychotics, situations that lead to changes in insulin signaling and extracellular levels of S100B. PMID:26875731

  3. Discovery of the First α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Antagonist Dependent upon Transmembrane AMPA Receptor Regulatory Protein (TARP) γ-8.

    PubMed

    Gardinier, Kevin M; Gernert, Douglas L; Porter, Warren J; Reel, Jon K; Ornstein, Paul L; Spinazze, Patrick; Stevens, F Craig; Hahn, Patric; Hollinshead, Sean P; Mayhugh, Daniel; Schkeryantz, Jeff; Khilevich, Albert; De Frutos, Oscar; Gleason, Scott D; Kato, Akihiko S; Luffer-Atlas, Debra; Desai, Prashant V; Swanson, Steven; Burris, Kevin D; Ding, Chunjin; Heinz, Beverly A; Need, Anne B; Barth, Vanessa N; Stephenson, Gregory A; Diseroad, Benjamin A; Woods, Tim A; Yu, Hong; Bredt, David; Witkin, Jeffrey M

    2016-05-26

    Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients. PMID:27067148

  4. Screening and Characterization of Hydrate Forms of T-3256336, a Novel Inhibitor of Apoptosis (IAP) Protein Antagonist.

    PubMed

    Takeuchi, Shoko; Kojima, Takashi; Hashimoto, Kentaro; Saito, Bunnai; Sumi, Hiroyuki; Ishikawa, Tomoyasu; Ikeda, Yukihiro

    2015-01-01

    Different crystal packing of hydrates from anhydrate crystals leads to different physical properties, such as solubility and stability. Investigation of the potential of varied hydrate formation, and understanding the stability in an anhydrous/hydrate system, are crucial to prevent an undesired transition during the manufacturing process and storage. Only one anhydrous form of T-3256336, a novel inhibitor of apoptosis (IAP) protein antagonist, was discovered during synthesis, and no hydrate form has been identified. In this study, we conducted hydrate screening such as dynamic water vapor sorption/desorption (DVS), and the slurry experiment, and characterized the solid-state properties of anhydrous/hydrate forms to determine the most desirable crystalline form for development. New hydrate forms, both mono-hydrate and hemi-hydrate forms, were discovered as a result of this hydrate screening. The characterization of two new hydrate forms was conducted, and the anhydrous form was determined to be the most desirable development form of T-3256336 in terms of solid-state stability. In addition, the stability of the anhydrous form was investigated using the water content and temperature controlled slurry experiment to obtain the desirable crystal form in the crystallization process. The water content regions of the stable phase of the desired form, the anhydrous form, were identified for the cooling crystallization process. PMID:26521850

  5. USP47 and C Terminus of Hsp70-Interacting Protein (CHIP) Antagonistically Regulate Katanin-p60-Mediated Axonal Growth

    PubMed Central

    Yang, Seung Wook; Oh, Kyu Hee; Park, Esther; Chang, Hyun Min; Park, Jung Mi; Seong, Min Woo; Ka, Seung Hyeun; Song, Woo Keun; Park, Dong Eun; Baas, Peter W.

    2013-01-01

    Katanin is a heterodimeric enzyme that severs and disassembles microtubules. While the p60 subunit has the enzyme activity, the p80 subunit regulates the p60 activity. The microtubule-severing activity of katanin plays an essential role in axonal growth. However, the mechanisms by which neuronal cells regulate the expression of katanin-p60 remains unknown. Here we showed that USP47 and C terminus of Hsp70-interacting protein (CHIP) antagonistically regulate the stability of katanin-p60 and thereby axonal growth. USP47 was identified as a katanin-p60-specific deubiquitinating enzyme for its stabilization. We also identified CHIP as a ubiquitin E3 ligase that promotes proteasome-mediated degradation of katanin-p60. Moreover, USP47 promoted axonal growth of cultured rat hippocampal neurons, whereas CHIP inhibited it. Significantly, treatment with basic fibroblast growth factor (bFGF), an inducer of axonal growth, increased the levels of USP47 and katanin-p60, but not CHIP. Consistently, bFGF treatment resulted in a marked decrease in the level of ubiquitinated katanin-p60 and thereby in the promotion of axonal growth. On the other hand, the level of USP47, but not CHIP, decreased concurrently with that of katanin-p60 as axons reached their target cells. These results indicate that USP47 plays a crucial role in the control of axonal growth during neuronal development by antagonizing CHIP-mediated katanin-p60 degradation. PMID:23904609

  6. Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function.

    PubMed

    Previs, Michael J; Mun, Ji Young; Michalek, Arthur J; Previs, Samantha Beck; Gulick, James; Robbins, Jeffrey; Warshaw, David M; Craig, Roger

    2016-03-22

    During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C's N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain's extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C's inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C's calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C's phosphorylation state. PMID:26908872

  7. Polycomb group protein bodybuilding: working out the routines.

    PubMed

    Sievers, Cem; Paro, Renato

    2013-09-30

    Polycomb group (PcG) proteins regulate gene expression by modifying chemical and structural properties of chromatin. Isono et al. (2013) now report in Developmental Cell a polymerization-dependent mechanism used by PcG proteins to form higher-order chromatin structures, referred to as Polycomb bodies, and demonstrate its necessity for gene silencing. PMID:24091008

  8. Four Tomato FLOWERING LOCUS T-Like Proteins Act Antagonistically to Regulate Floral Initiation.

    PubMed

    Cao, Kai; Cui, Lirong; Zhou, Xiaoting; Ye, Lin; Zou, Zhirong; Deng, Shulin

    2015-01-01

    The transition from vegetative growth to floral meristems in higher plants is regulated through the integration of internal cues and environmental signals. We were interested to examine the molecular mechanism of flowering in the day-neutral plant tomato (Solanum lycopersicum L.) and the effect of environmental conditions on tomato flowering. Analysis of the tomato genome uncovered 13 PEBP (phosphatidylethanolamine-binding protein) genes, and found six of them were FT-like genes which named as SlSP3D, SlSP6A, SlSP5G, SlSP5G1, SlSP5G2, and SlSP5G3. Six FT-like genes were analyzed to clarify their functional roles in flowering using transgenic and expression analyses. We found that SlSP5G, SlSP5G2, and SlSP5G3 proteins were floral inhibitors whereas only SlSP3D/SFT (SINGLE FLOWER TRUSS) was a floral inducer. SlSP5G was expressed at higher levels in long day (LD) conditions compared to short day (SD) conditions while SlSP5G2 and SlSP5G3 showed the opposite expression patterns. The silencing of SlSP5G by VIGS (Virus induced gene silencing) resulted in tomato plants that flowered early under LD conditions and the silencing of SlSP5G2 and SlSP5G3 led to early flowering under SD conditions. The higher expression levels of SlSP5G under LD conditions were not seen in phyB1 mutants, and the expression levels of SlSP5G2 and SlSP5G3 were increased in phyB1 mutants under both SD and LD conditions compared to wild type plants. These data suggest that SlSP5G, SlSP5G2, and SlSP5G3 are controlled by photoperiod, and the different expression patterns of FT-like genes under different photoperiod may contribute to tomato being a day neutral plant. In addition, PHYB1 mediate the expression of SlSP5G, SlSP5G2, and SlSP5G3 to regulate flowering in tomato. PMID:26793202

  9. Four Tomato FLOWERING LOCUS T-Like Proteins Act Antagonistically to Regulate Floral Initiation

    PubMed Central

    Cao, Kai; Cui, Lirong; Zhou, Xiaoting; Ye, Lin; Zou, Zhirong; Deng, Shulin

    2016-01-01

    The transition from vegetative growth to floral meristems in higher plants is regulated through the integration of internal cues and environmental signals. We were interested to examine the molecular mechanism of flowering in the day-neutral plant tomato (Solanum lycopersicum L.) and the effect of environmental conditions on tomato flowering. Analysis of the tomato genome uncovered 13 PEBP (phosphatidylethanolamine-binding protein) genes, and found six of them were FT-like genes which named as SlSP3D, SlSP6A, SlSP5G, SlSP5G1, SlSP5G2, and SlSP5G3. Six FT-like genes were analyzed to clarify their functional roles in flowering using transgenic and expression analyses. We found that SlSP5G, SlSP5G2, and SlSP5G3 proteins were floral inhibitors whereas only SlSP3D/SFT (SINGLE FLOWER TRUSS) was a floral inducer. SlSP5G was expressed at higher levels in long day (LD) conditions compared to short day (SD) conditions while SlSP5G2 and SlSP5G3 showed the opposite expression patterns. The silencing of SlSP5G by VIGS (Virus induced gene silencing) resulted in tomato plants that flowered early under LD conditions and the silencing of SlSP5G2 and SlSP5G3 led to early flowering under SD conditions. The higher expression levels of SlSP5G under LD conditions were not seen in phyB1 mutants, and the expression levels of SlSP5G2 and SlSP5G3 were increased in phyB1 mutants under both SD and LD conditions compared to wild type plants. These data suggest that SlSP5G, SlSP5G2, and SlSP5G3 are controlled by photoperiod, and the different expression patterns of FT-like genes under different photoperiod may contribute to tomato being a day neutral plant. In addition, PHYB1 mediate the expression of SlSP5G, SlSP5G2, and SlSP5G3 to regulate flowering in tomato. PMID:26793202

  10. Epigenetic inactivation of the canonical Wnt antagonist secreted frizzled-related protein 1 in hepatocellular carcinoma cells.

    PubMed

    Wu, Y; Li, J; Sun, C Y; Zhou, Y; Zhao, Y F; Zhang, S J

    2012-01-01

    Secreted Frizzled-related protein 1 (sFRP1), as one of most important Wnt antagonists, is frequently silenced by promoter hypermethylation in many types of tumor, including hepatocellular carcinoma (HCC). In this study, we aimed to investigate whether restoration of sFRP1 affected HCC metastatic behavior. sFRP1 mRNA expression and promoter methylation in HCC tissues and cell lines were examined using RT-PCR and methylation-specific PCR (MS-PCR), respectively. sFRP1 protein expression was assessed by Western Blot. We generated stable HCC cell line restoration of sFRP1 in HepG2 cells, which naturally do not express detectable sFRP1 mRNA. The effects of exogenous sFRP1 on HepG2 cell invasion were investigated using trans-well assay. Also the effects of sFRP1 re-expression on the β-catenin/T-cell factor-dependent transcription activity was measured by luciferase assay.sFRP1 promoter methylation was frequently observed in HCC tissues (60%) and cell lines (75%). All samples with sFRP1 methylation showed down-regulation of sFRP1 expression in HCC cell lines. Demethylation treatment with 5-aza-20-deoxycytidine in HCC cells restored sFRP1 expression. Restoration of sFRP1 substantially impaired the invasive potentials of HepG2 cells. Moreover, exogenous sFRP1 caused significant decrease of β-catenin/T-cell factor-dependent transcription activity.These findings demonstrate that sFRP1 silencing due to promoter hypermethylation is a major event during tumorigenesis. sFRP1 is also a negative modulator of canonical Wnt signaling, which could contribute to metastasis in HCC progression, thus providing a possible therapeutic strategy against HCC. PMID:22296502

  11. The protein interaction landscape of the human CMGC kinase group.

    PubMed

    Varjosalo, Markku; Keskitalo, Salla; Van Drogen, Audrey; Nurkkala, Helka; Vichalkovski, Anton; Aebersold, Ruedi; Gstaiger, Matthias

    2013-04-25

    Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways. PMID:23602568

  12. Protein kinase C-alpha and -beta play antagonistic roles in the differentiation process of THP-1 cells.

    PubMed

    Dieter, P; Schwende, H

    2000-05-01

    The roles of protein kinase C (PKC) isoenzymes in the differentiation process of THP-1 cells are investigated. Inhibition of PKC by RO 31-8220 reduces the phagocytosis of latex particles and the release of superoxide, prostaglandin E(2) (PGE(2)), and tumour necrosis factor (TNF)-alpha. The proliferation of THP-1 cells is slightly enhanced by RO 31-8220. Stable transfection of THP-1 cells with asPKC-alpha, and incubation of THP-1 cells with antisense (as) PKC-alpha oligodeoxynucleotides reduces PKC-alpha levels and PKC activity. asPKC-alpha-transfected THP-1 cells show a decreased phagocytosis and a decreased release of superoxide, PGE(2) and TNF-alpha. The proliferation of asPKC-alpha-transfected THP-1 cells is enhanced. Stable transfection of THP-1 cells with asPKC-beta, and incubation of THP-1 cells with asPKC-beta oligodeoxynucleotides, reduces PKC-beta levels and PKC activity. asPKC-beta-transfected THP-1 cells show a decreased phagocytosis, a decreased TNF-alpha release, and a decreased proliferation. However, no difference is measured in the release of superoxide and PGE(2). These results suggest that: (1) PKC-alpha but not PKC-beta is involved in the release of superoxide and PGE(2); (2) TNF-alpha release and the phagocytosis of latex particles are mediated by PKC-alpha, PKC-beta, and other PKC isoenzymes; and (3) PKC-alpha and PKC-beta play antagonistic roles in the differentiation process of THP-1 cells. PKC-alpha promotes the differentiation process of THP-1 cells, PKC-beta retards the differentiation of THP-1 cells into macrophage-like cells. PMID:10822170

  13. Classifying proteins into functional groups based on all-versus-all BLAST of 10 million proteins.

    PubMed

    Kolker, Natali; Higdon, Roger; Broomall, William; Stanberry, Larissa; Welch, Dean; Lu, Wei; Haynes, Winston; Barga, Roger; Kolker, Eugene

    2011-01-01

    To address the monumental challenge of assigning function to millions of sequenced proteins, we completed the first of a kind all-versus-all sequence alignments using BLAST for 9.9 million proteins in the UniRef100 database. Microsoft Windows Azure produced over 3 billion filtered records in 6 days using 475 eight-core virtual machines. Protein classification into functional groups was then performed using Hive and custom jars implemented on top of Apache Hadoop utilizing the MapReduce paradigm. First, using the Clusters of Orthologous Genes (COG) database, a length normalized bit score (LNBS) was determined to be the best similarity measure for classification of proteins. LNBS achieved sensitivity and specificity of 98% each. Second, out of 5.1 million bacterial proteins, about two-thirds were assigned to significantly extended COG groups, encompassing 30 times more assigned proteins. Third, the remaining proteins were classified into protein functional groups using an innovative implementation of a single-linkage algorithm on an in-house Hadoop compute cluster. This implementation significantly reduces the run time for nonindexed queries and optimizes efficient clustering on a large scale. The performance was also verified on Amazon Elastic MapReduce. This clustering assigned nearly 2 million proteins to approximately half a million different functional groups. A similar approach was applied to classify 2.8 million eukaryotic sequences resulting in over 1 million proteins being assign to existing KOG groups and the remainder clustered into 100,000 functional groups. PMID:21809957

  14. Antagonistic regulation of swelling-activated Cl− current in rabbit ventricle by Src and EGFR protein tyrosine kinases

    PubMed Central

    Ren, Zuojun; Baumgarten, Clive M.

    2005-01-01

    Regulation of swelling-activated Cl− current (ICl,swell) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates ICl,swell and to identify the PTK involved, we studied the effects of a broad-spectrum PTK inhibitor (genistein), selective inhibitors of Src (PP2, a pyrazolopyrimidine) and epidermal growth factor receptor (EGFR) kinase (PD-153035), and a protein tyrosine phosphatase (PTP) inhibitor (orthovanadate). ICl,swell evoked by hyposmotic swelling was increased 181 ± 17% by 100 μM genistein, and the genistein-induced current was blocked by the selective ICl,swell blocker tamoxifen (10 μM). Block of Src with PP2 (10 μM) stimulated tamoxifen-sensitive ICl,swell by 234 ± 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 μM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited ICl,swell and prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited ICl,swell. Several lines of evidence argue that the PP2-stimulated current was ICl,swell: 1) the stimulation was volume dependent, 2) the current was blocked by tamoxifen, 3) the current outwardly rectified with both symmetrical and physiological Cl− gradients, and 4) the current reversed near the Cl− equilibrium potential. To rule out contributions of other currents, Cd2+ (0.2 mM) and Ba2+ (1 mM) were added to the bath. Surprisingly, Cd2+ suppressed the decay of Cd2+ plus Ba2+ eliminated time-dependent ICl,swell, and currents between −100 and −100 mV. Nevertheless, these divalent ions did not eliminate ICl,swell or prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls ICl,swell, and regulation of ICl,swell by the Src and EGFR kinase families of PTK is antagonistic. PMID:15681694

  15. TRPV1 Antagonists Elevate Cell Surface Populations of Receptor Protein and Exacerbate TRPV1-Mediated Toxicities in Human Lung Epithelial Cells

    PubMed Central

    Johansen, Mark E.; Reilly, Christopher A.; Yost, Garold S.

    2008-01-01

    TRPV1 mediates cell death and pro-inflammatory cytokine production in lung epithelial cells exposed to prototypical receptor agonists. This study shows that NHBE, BEAS-2B and TRPV1 over-expressing BEAS-2B cells pre-treated with various TRPV1 antagonists become sensitized to the prototypical TRPV1 agonist, nonivamide, via a mechanism that involves translocation of existing receptor from the endoplasmic reticulum to the plasma membrane. As such, typical cellular responses to agonist treatment, as measured by calcium flux, inflammatory cytokine gene induction, and cytotoxicity were exacerbated. These data were in contrast to the results obtained when TRPV1 antagonists were co-administered with nonivamide; conditions which inhibited TRPV1-mediated effects. The antagonists LJO-328, SC0030, and capsazepine increased the cytotoxicity of nonivamide by ~20-fold and agonist-induced calcium flux by ~6-fold. Inflammatory-cytokine gene induction by nonivamide was also increased significantly by pre-treatment with the antagonists. The enhanced responses were inhibited by the co-administration of antagonists with nonivamide, confirming that increases in sensitivity were attributable to increased TRPV1-associated activity. Sensitization was attenuated by brefeldin A (a golgi transport inhibitor), but not cycloheximide (a protein synthesis inhibitor), or actinomycin D (a transcription inhibitor). Sensitized cells exhibited increased calcium flux from extracellular calcium sources, while unsensitized cells exhibited calcium flux originating primarily from intracellular stores. These results demonstrate the presence of a novel mechanism for regulating the sub-cellular distribution of TRPV1 and subsequent control of cellular sensitivity to TRPV1 agonists. PMID:16120755

  16. The TRIM-NHL Protein LIN-41 and the OMA RNA-Binding Proteins Antagonistically Control the Prophase-to-Metaphase Transition and Growth of Caenorhabditis elegans Oocytes

    PubMed Central

    Spike, Caroline A.; Coetzee, Donna; Eichten, Carly; Wang, Xin; Hansen, Dave; Greenstein, David

    2014-01-01

    In many animals, oocytes enter meiosis early in their development but arrest in meiotic prophase I. Oocyte growth, which occurs during this arrest period, enables the acquisition of meiotic competence and the capacity to produce healthy progeny. Meiotic resumption, or meiotic maturation, involves the transition to metaphase I (M phase) and is regulated by intercellular signaling and cyclin-dependent kinase activation. Premature meiotic maturation would be predicted to diminish fertility as the timing of this event, which normally occurs after oocyte growth is complete, is crucial. In the accompanying article in this issue, we identify the highly conserved TRIM-NHL protein LIN-41 as a translational repressor that copurifies with OMA-1 and OMA-2, RNA-binding proteins redundantly required for normal oocyte growth and meiotic maturation. In this article, we show that LIN-41 enables the production of high-quality oocytes and plays an essential role in controlling and coordinating oocyte growth and meiotic maturation. lin-41 null mutants display a striking defect that is specific to oogenesis: pachytene-stage cells cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, assemble spindles, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 appears to contribute to premature M-phase entry in lin-41 mutant oocytes. Genetic and phenotypic analyses indicate that LIN-41 and OMA-1/2 exhibit an antagonistic relationship, and we suggest that translational regulation by these proteins could be important for controlling and coordinating oocyte growth and meiotic maturation. PMID:25261698

  17. The TRIM-NHL protein LIN-41 and the OMA RNA-binding proteins antagonistically control the prophase-to-metaphase transition and growth of Caenorhabditis elegans oocytes.

    PubMed

    Spike, Caroline A; Coetzee, Donna; Eichten, Carly; Wang, Xin; Hansen, Dave; Greenstein, David

    2014-12-01

    In many animals, oocytes enter meiosis early in their development but arrest in meiotic prophase I. Oocyte growth, which occurs during this arrest period, enables the acquisition of meiotic competence and the capacity to produce healthy progeny. Meiotic resumption, or meiotic maturation, involves the transition to metaphase I (M phase) and is regulated by intercellular signaling and cyclin-dependent kinase activation. Premature meiotic maturation would be predicted to diminish fertility as the timing of this event, which normally occurs after oocyte growth is complete, is crucial. In the accompanying article in this issue, we identify the highly conserved TRIM-NHL protein LIN-41 as a translational repressor that copurifies with OMA-1 and OMA-2, RNA-binding proteins redundantly required for normal oocyte growth and meiotic maturation. In this article, we show that LIN-41 enables the production of high-quality oocytes and plays an essential role in controlling and coordinating oocyte growth and meiotic maturation. lin-41 null mutants display a striking defect that is specific to oogenesis: pachytene-stage cells cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, assemble spindles, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 appears to contribute to premature M-phase entry in lin-41 mutant oocytes. Genetic and phenotypic analyses indicate that LIN-41 and OMA-1/2 exhibit an antagonistic relationship, and we suggest that translational regulation by these proteins could be important for controlling and coordinating oocyte growth and meiotic maturation. PMID:25261698

  18. Genetic Incorporation of a Reactive Isothiocyanate Group into Proteins.

    PubMed

    Xuan, Weimin; Li, Jack; Luo, Xiaozhou; Schultz, Peter G

    2016-08-16

    Methods for the site-specific modification of proteins are useful for introducing biological probes into proteins and engineering proteins with novel activities. Herein, we genetically encode a novel noncanonical amino acid (ncAA) that contains an aryl isothiocyanate group which can form stable thiourea crosslinks with amines under mild conditions. We show that this ncAA (pNCSF) allows the selective conjugation of proteins to amine-containing molecular probes through formation of a thiourea bridge. pNCSF was also used to replace a native salt bridge in myoglobin with an intramolecular crosslink to a proximal Lys residue, leading to increased thermal stability. Finally, we show that pNCSF can form stable intermolecular crosslinks between two interacting proteins. PMID:27418387

  19. Design, Synthesis, and Evaluation of Nonretinoid Retinol Binding Protein 4 Antagonists for the Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease

    PubMed Central

    2015-01-01

    Accumulation of lipofuscin in the retina is associated with pathogenesis of atrophic age-related macular degeneration and Stargardt disease. Lipofuscin bisretinoids (exemplified by N-retinylidene-N-retinylethanolamine) seem to mediate lipofuscin toxicity. Synthesis of lipofuscin bisretinoids depends on the influx of retinol from serum to the retina. Compounds antagonizing the retinol-dependent interaction of retinol-binding protein 4 (RBP4) with transthyretin in the serum would reduce serum RBP4 and retinol and inhibit bisretinoid formation. We recently showed that A1120 (3), a potent carboxylic acid based RBP4 antagonist, can significantly reduce lipofuscin bisretinoid formation in the retinas of Abca4–/– mice. As part of the NIH Blueprint Neurotherapeutics Network project we undertook the in vitro exploration to identify novel conformationally flexible and constrained RBP4 antagonists with improved potency and metabolic stability. We also demonstrate that upon acute and chronic dosing in rats, 43, a potent cyclopentyl fused pyrrolidine antagonist, reduced circulating plasma RBP4 protein levels by approximately 60%. PMID:25210858

  20. The zipper groups of the amyloid state of proteins

    SciTech Connect

    Stroud, James C.

    2013-04-01

    A formal derivation is provided of the 15 symmetry groups (zipper groups) available to the amyloid homosteric zipper. Fibrous proteins in the amyloid state are found both associated with numerous diseases and in the normal functions of cells. Amyloid fibers contain a repetitive spine, commonly built from a pair of β-sheets whose β-strands run perpendicular to the fiber direction and whose side chains interdigitate, much like the teeth of a zipper. In fiber spines known as homosteric zippers, identical protein segments sharing identical packing environments make the two β-sheets. In previous work based on atomic resolution crystal structures of homosteric zippers derived from a dozen proteins, the symmetries of homosteric zippers were categorized into eight classes. Here, it is shown through a formal derivation that each homosteric zipper class corresponds to a unique set of symmetry groups termed ‘zipper groups’. Furthermore, the eight previously identified classes do not account for all of the 15 possible zipper groups, which may be categorized into the complete set of ten classes. Because of their foundations in group theory, the 15 zipper groups provide a mathematically rigorous classification for homosteric zippers.

  1. Banana Ovate Family Protein MaOFP1 and MADS-Box Protein MuMADS1 Antagonistically Regulated Banana Fruit Ripening

    PubMed Central

    Hu, Wei; Miao, Hongxia; Zhang, Jianbin; Jia, Caihong; Wang, Zhuo; Xu, Biyu; Jin, Zhiqiang

    2015-01-01

    The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening. PMID:25886169

  2. Banana Ovate family protein MaOFP1 and MADS-box protein MuMADS1 antagonistically regulated banana fruit ripening.

    PubMed

    Liu, Juhua; Zhang, Jing; Hu, Wei; Miao, Hongxia; Zhang, Jianbin; Jia, Caihong; Wang, Zhuo; Xu, Biyu; Jin, Zhiqiang

    2015-01-01

    The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening. PMID:25886169

  3. Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown.

    PubMed Central

    Hershko, A; Heller, H; Eytan, E; Kaklij, G; Rose, I A

    1984-01-01

    Previous studies suggest that the conjugation of ubiquitin to NH2 groups of proteins is required for protein breakdown. We now show that the selective modification of NH2-terminal alpha-NH2 groups of globin and lysozyme prevents their degradation by the ubiquitin proteolytic system from reticulocytes. The conjugation by ubiquitin of epsilon-NH2 groups of lysine residues, usually seen in multiples, was also inhibited in alpha-NH2-blocked proteins. Naturally occurring N alpha-acetylated proteins are not degraded by the ubiquitin system at a significant rate, while their nonacetylated counterparts from other species are good substrates. This suggests that one function of N alpha-acetylation of cellular proteins is to prevent their degradation by the ubiquitin system. alpha-NH2-blocked proteins can have their activity as substrates for degradation increased by incorporation of alpha-NH2 groups through the introduction of polyalanine side chains. Proteins in which most epsilon-NH2 groups are blocked but the alpha-NH2 group is free are degraded by the ubiquitin system, but at a reduced rate. It is therefore suggested that the exposure of a free NH2 terminus of proteins is required for degradation and probably initiates the formation of ubiquitin conjugates committed for degradation. Images PMID:6095265

  4. The Meat and Protein Group. The Food Guide Pyramid.

    ERIC Educational Resources Information Center

    Frost, Helen

    This booklet for young children is part of a series that supports national science standards related to physical health and nutrition, describing and illustrating the importance of using the Food Guide Pyramid and eating from the meat and protein group. Colorful photographs support early readers in understanding the text. The repetition of words…

  5. pK values of the ionizable groups of proteins.

    PubMed

    Thurlkill, Richard L; Grimsley, Gerald R; Scholtz, J Martin; Pace, C Nick

    2006-05-01

    We have used potentiometric titrations to measure the pK values of the ionizable groups of proteins in alanine pentapeptides with appropriately blocked termini. These pentapeptides provide an improved model for the pK values of the ionizable groups in proteins. Our pK values determined in 0.1 M KCl at 25 degrees C are: 3.67+/-0.03 (alpha-carboxyl), 3.67+/-0.04 (Asp), 4.25+/-0.05 (Glu), 6.54+/-0.04 (His), 8.00+/-0.03 (alpha-amino), 8.55+/-0.03 (Cys), 9.84+/-0.11 (Tyr), and 10.40+/-0.08 (Lys). The pK values of some groups differ from the Nozaki and Tanford (N & T) pK values often used in the literature: Asp (3.67 this work vs. 4.0 N & T); His (6.54 this work vs. 6.3 N & T); alpha-amino (8.00 this work vs. 7.5 N & T); Cys (8.55 this work vs. 9.5 N & T); and Tyr (9.84 this work vs. 9.6 N & T). Our pK values will be useful to those who study pK perturbations in folded and unfolded proteins, and to those who use theory to gain a better understanding of the factors that determine the pK values of the ionizable groups of proteins. PMID:16597822

  6. Behaviour of protein carbonyl groups in juvenile myocardial infarction.

    PubMed

    Caimi, Gregorio; Canino, Baldassare; Incalcaterra, Egle; Ferrera, Eleonora; Montana, Maria; Lo Presti, Rosalia

    2013-01-01

    Acute myocardial infarction (AMI) is accompanied by oxidative stress, and protein oxidation is among the consequences of oxidative stress. We examined the plasma concentration of protein carbonyl groups (PC), a marker of protein oxidation, in a group of young subjects with AMI (45 men and 5 women; mean age 40.4 ± 4.8 yrs). We found a significant increase of PC (p < 0.001) in comparison with normal controls. No difference was observed between patients with AMI characterized by elevated ST segment and those without elevation of ST segment. There was no correlation between the ejection fraction and PC in the whole group nor in the subgroups of STEMI and non-STEMI patients. Subdividing the whole group of AMI patients according to the number of risk factors and the number of stenosed coronary vessels, the difference in PC level was not statistically significant among the subgroups. This study showed an increased protein oxidation in young subjects with recent AMI. Further investigation is needed to ascertain whether this can be a target of therapeutic intervention. PMID:22504219

  7. A potent and orally active antagonist of multiple inhibitor of apoptosis proteins (IAPs) (SM-406/AT-406) in clinical development for cancer treatment

    PubMed Central

    Cai, Qian; Sun, Haiying; Peng, Yuefeng; Lu, Jianfeng; Nikolovska-Coleska, Zaneta; McEachern, Donna; Liu, Liu; Qiu, Su; Yang, Chao-Yie; Miller, Rebecca; Yi, Han; Zhang, Tao; Sun, Duxin; Kang, Sanmao; Guo, Ming; Leopold, Lance; Yang, Dajun; Wang, Shaomeng

    2011-01-01

    We report the discovery and characterization of SM-406 (compound 2), a potent and orally bioavailable Smac mimetic and an antagonist of the inhibitor of apoptosis proteins (IAPs). This compound binds to XIAP, cIAP1 and cIAP2 proteins with Ki values of 66.4 nM, 1.9 nM and 5.1 nM, respectively. Compound 2 effectively antagonizes XIAP BIR3 protein in a cell-free functional assay, induces rapid degradation of cellular cIAP1 protein and inhibits cancer cell growth in various human cancer cell lines. It has good oral bioavailability in mice, rats, non-human primates and dogs, is highly effective in induction of apoptosis in xenograft tumors and is capable of complete inhibition of tumor growth. Compound 2 is currently in Phase I clinical trials for the treatment of human cancer. PMID:21443232

  8. Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Antagonistically Regulate Fission Yeast fbp1 Transcription by Employing Different Modes of Action at Two Upstream Activation Sites

    PubMed Central

    Neely, Lori A.; Hoffman, Charles S.

    2000-01-01

    A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of the Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized two cis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulate fbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site. PMID:10938120

  9. The Antagonistic Effect of Selenium on Lead-Induced Inflammatory Factors and Heat Shock Proteins mRNA Expression in Chicken Livers.

    PubMed

    Wang, Hao; Li, Shu; Teng, Xiaohua

    2016-06-01

    The aim of this study was to investigate the effect of lead (Pb) poisoning on nitric oxide (NO) content, inducible nitric oxide synthase (iNOS) activity, the messenger RNA (mRNA) levels of inflammatory factors (nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), prostaglandin E synthases (PTGEs), and iNOS), heat shock proteins (HSPs) (HSP27, HSP40, HSP60, HSP70, and HSP90), and the antagonistic effect of selenium (Se) on Pb in chicken livers. One hundred eighty 7-day-old male chickens were randomly divided into four groups and were fed commercial diet and drinking water, Na2SeO3-added commercial diet and drinking water, commercial diet and (CH3OO)2Pb-added drinking water, and Na2SeO3-added commercial diet and (CH3OO)2Pb-added drinking water, respectively, for 30, 60, and 90 days. Then, NO content, iNOS activity, and the mRNA levels of NF-κB, TNF-α, COX-2, PTGEs, iNOS, HSP27, HSP40, HSP60, HSP70, and HSP90 were examined in chicken livers. The results showed that Pb poisoning induced NO content, iNOS activity, and mRNA expression of inflammation factors and HSPs in chicken livers. In addition, Se alleviated Pb-induced increase of inflammation factor and HSP expression in chicken livers. PMID:26470710

  10. Xeroderma pigmentosum group A correcting protein from calf thymus.

    PubMed

    Eker, A P; Vermeulen, W; Miura, N; Tanaka, K; Jaspers, N G; Hoeijmakers, J H; Bootsma, D

    1992-09-01

    A proteinous factor was purified from calf thymus and HeLa cells, which specifically corrects the excision repair defect of xeroderma pigmentosum complementation group A (XP-A) cells. Recovery of UV-induced unscheduled DNA synthesis after microinjection of XP-A cells was used as a quantitative assay for the correcting activity of protein preparations. XP-A correcting protein appears to be very stable as it withstands heating to 100 degrees C and treatment with SDS or 6 M urea. A molecular weight of 40-45 kD was found both under native (gel filtration) and denaturing (SDS-PAGE) conditions. Calf XP-A protein binds to single-stranded DNA more strongly than to double-stranded DNA, but shows no clear preference for UV-irradiated DNA. Polyclonal antibodies raised against human recombinant XP-A protein, which strongly inhibit UV-induced unscheduled DNA synthesis of normal human cells, completely abolished XP-A correcting activity when mixed with calf thymus preparations. This indicates a close relationship between human gene product and the calf protein. In the final preparation two main protein bands were present. Only one band at approx. 41 kD showed both DNA binding activity in Southwestern blots and immune reaction with human XP-A antibody, suggesting that this is the active calf XP-A correcting factor. PMID:1380654

  11. Chromatin topology is coupled to Polycomb group protein subnuclear organization

    PubMed Central

    Wani, Ajazul H.; Boettiger, Alistair N.; Schorderet, Patrick; Ergun, Ayla; Münger, Christine; Sadreyev, Ruslan I.; Zhuang, Xiaowei; Kingston, Robert E.; Francis, Nicole J.

    2016-01-01

    The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions. PMID:26759081

  12. Chromatin topology is coupled to Polycomb group protein subnuclear organization.

    PubMed

    Wani, Ajazul H; Boettiger, Alistair N; Schorderet, Patrick; Ergun, Ayla; Münger, Christine; Sadreyev, Ruslan I; Zhuang, Xiaowei; Kingston, Robert E; Francis, Nicole J

    2016-01-01

    The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions. PMID:26759081

  13. Bicyclic [3.3.0]-Octahydrocyclopenta[c]pyrrolo Antagonists of Retinol Binding Protein 4: Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease.

    PubMed

    Cioffi, Christopher L; Racz, Boglarka; Freeman, Emily E; Conlon, Michael P; Chen, Ping; Stafford, Douglas G; Schwarz, Daniel M C; Zhu, Lei; Kitchen, Douglas B; Barnes, Keith D; Dobri, Nicoleta; Michelotti, Enrique; Cywin, Charles L; Martin, William H; Pearson, Paul G; Johnson, Graham; Petrukhin, Konstantin

    2015-08-13

    Antagonists of retinol-binding protein 4 (RBP4) impede ocular uptake of serum all-trans retinol (1) and have been shown to reduce cytotoxic bisretinoid formation in the retinal pigment epithelium (RPE), which is associated with the pathogenesis of both dry age-related macular degeneration (AMD) and Stargardt disease. Thus, these agents show promise as a potential pharmacotherapy by which to stem further neurodegeneration and concomitant vision loss associated with geographic atrophy of the macula. We previously disclosed the discovery of a novel series of nonretinoid RBP4 antagonists, represented by bicyclic [3.3.0]-octahydrocyclopenta[c]pyrrolo analogue 4. We describe herein the utilization of a pyrimidine-4-carboxylic acid fragment as a suitable isostere for the anthranilic acid appendage of 4, which led to the discovery of standout antagonist 33. Analogue 33 possesses exquisite in vitro RBP4 binding affinity and favorable drug-like characteristics and was found to reduce circulating plasma RBP4 levels in vivo in a robust manner (>90%). PMID:26181715

  14. The Antagonistic Effect of Selenium on Lead-Induced Inflammatory Factors and Heat Shock Protein mRNA Level in Chicken Cartilage Tissue.

    PubMed

    Zheng, Shufang; Song, Huanyu; Gao, Han; Liu, Chunpeng; Zhang, Ziwei; Fu, Jing

    2016-09-01

    Selenium (Se) is recognized as a necessary trace mineral in animal diets, including those of birds. Lead (Pb) is a toxic heavy metal and can damage organs in humans and animals. Complex antagonistic interactions between Se and heavy metals have been reported in previous studies. However, little is known regarding the effects of Se on Pb-induced toxicity and the expression of inflammatory factors and heat shock proteins (HSPs) in the cartilage of chickens. In this present study, we fed chickens either with Se or Pb or both Se and Pb supplement and later analyzed the mRNA expressions of inflammatory factors (inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2)) and HSPs (Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90). The results showed that Se and Pb influenced the expression of inflammatory factors and HSP genes in the chicken cartilage tissues. Additionally, we also found that antagonistic interaction existed between Se and Pb supplementation. Our findings suggested that Se could exert a antagonistic effect on Pb in chicken cartilage tissues. PMID:26831653

  15. A Gβ protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes

    PubMed Central

    Janganan, Thamarai K.; Chen, Gongyou; Chen, Daliang; Menino, João F.; Rodrigues, Fernando; Borges-Walmsley, Maria I.; Walmsley, Adrian R.

    2015-01-01

    The human pathogenic fungus Paracoccidioides brasiliensis (Pb) undergoes a morphological transition from a saprobic mycelium to pathogenic yeast that is controlled by the cAMP-signaling pathway. There is a change in the expression of the Gβ-protein PbGpb1, which interacts with adenylate cyclase, during this morphological transition. We exploited the fact that the cAMP-signaling pathway of Saccharomyces cerevisiae does not include a Gβ-protein to probe the functional role of PbGpb1. We present data that indicates that PbGpb1 and the transcriptional regulator PbTupA both bind to the PKA protein PbTpk2. PbTPK2 was able to complement a TPK2Δ strain of S. cerevisiae, XPY5a/α, which was defective in pseudohyphal growth. Whilst PbGPB1 had no effect on the parent S. cerevisiae strain, MLY61a/α, it repressed the filamentous growth of XPY5a/α transformed with PbTPK2, behaviour that correlated with a reduced expression of the floculin FLO11. In vitro, PbGpb1 reduced the kinase activity of PbTpk2, suggesting that inhibition of PbTpk2 by PbGpb1 reduces the level of expression of Flo11, antagonizing the filamentous growth of the cells. In contrast, expressing the co-regulator PbTUPA in XPY5a/α cells transformed with PbTPK2, but not untransformed cells, induced hyperfilamentous growth, which could be antagonized by co-transforming the cells with PbGPB1. PbTUPA was unable to induce the hyperfilamentous growth of a FLO8Δ strain, suggesting that PbTupA functions in conjunction with the transcription factor Flo8 to control Flo11 expression. Our data indicates that P. brasiliensis PbGpb1 and PbTupA, both of which have WD/β-propeller structures, bind to PbTpk2 to act as antagonistic molecular switches of cell morphology, with PbTupA and PbGpb1 inducing and repressing filamentous growth, respectively. Our findings define a potential mechanism for controlling the morphological switch that underpins the virulence of dimorphic fungi. PMID:26334875

  16. Characterization of a stress protein from group B Neisseria meningitidis.

    PubMed Central

    Arakere, G; Kessel, M; Nguyen, N; Frasch, C E

    1993-01-01

    Increased levels of a 65-kDa stress protein (Msp65) were observed in group B Neisseria meningitidis grown under stationary-growth conditions. Electron microscopy showed two apposing rings of seven subunits, a structure typical of Escherichia coli GroEL. Msp65 was not found in either the periplasmic space or the outer membrane. Several important differences between the GroEL analogs of N. meningitidis and Neisseria gonorrhoeae are discussed. Images PMID:8099073

  17. Angiotensin II type 1 receptor antagonists inhibit basal as well as low-density lipoprotein and platelet-activating factor-stimulated human monocyte chemoattractant protein-1.

    PubMed

    Proudfoot, Julie M; Croft, Kevin D; Puddey, Ian B; Beilin, Lawrence J

    2003-06-01

    Monocyte chemoattractant protein-1 (MCP-1) is a potent chemotactic agent for monocytes and other cells and is thought to be involved in atherosclerosis, recruiting monocytes to the subendothelial space or to the site of inflammation. Angiotensin II has been demonstrated, at least in animal models, to stimulate MCP-1 expression. We investigated the effect of the angiotensin II type 1 (AT1) receptor antagonists irbesartan and losartan on MCP-1 production by freshly isolated human monocytes. Irbesartan and losartan inhibited basal MCP-1 production in a dose-dependent manner. Low-density lipoprotein (LDL) stimulated MCP-1 in a concentration-dependent manner, with 200 microg/ml LDL protein giving a 2-fold increase in MCP-1. Irbesartan and losartan dose dependently blocked LDL-stimulated MCP-1. An angiotensin II type 2 receptor antagonist, S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid (PD123319), had no significant effect on basal MCP-1 levels or LDL-stimulated MCP-1. After noting homology between the AT1 receptor and the platelet-activating factor (PAF) receptor, we showed that irbesartan inhibited both [3H]PAF binding to human monocytes and carbamyl-PAF stimulation of MCP-1. However, irbesartan affinity for the PAF receptor was 700 times less than PAF, suggesting that there may be another mechanism for irbesartan inhibition of PAF-stimulated MCP-1. This is the first report showing that AT1 receptor antagonists inhibit basal as well as LDL- and PAF-stimulated MCP-1 production in freshly isolated human monocytes. PMID:12626661

  18. Rapid, Structure-Based Exploration of Pipecolic Acid Amides as Novel Selective Antagonists of the FK506-Binding Protein 51.

    PubMed

    Gaali, Steffen; Feng, Xixi; Hähle, Andreas; Sippel, Claudia; Bracher, Andreas; Hausch, Felix

    2016-03-24

    The FK506-binding protein 51 (FKBP51) is a key regulator of stress hormone receptors and an established risk factor for stress-related disorders. Drug development for FKBP51 has been impaired by the structurally similar but functionally opposing homologue FKBP52. High selectivity between FKBP51 and FKBP52 can be achieved by ligands that stabilize a recently discovered FKBP51-favoring conformation. However, drug-like parameters for these ligands remained unfavorable. In the present study, we replaced the potentially labile pipecolic ester group of previous FKBP51 ligands by various low molecular weight amides. This resulted in the first series of pipecolic acid amides, which had much lower molecular weights without affecting FKBP51 selectivity. We discovered a geminally substituted cyclopentyl amide as a preferred FKBP51-binding motif and elucidated its binding mode to provide a new lead structure for future drug optimization. PMID:26954324

  19. IL-1 receptor antagonist affects the plasma protein response of Hep 3B cells to conditioned medium from lipopolysaccharide-stimulated monocytes.

    PubMed

    Damtew, B; Rzewnicki, D; Lozanski, G; Kushner, I

    1993-05-01

    The availability of the IL-1R antagonist (IL-1ra) has made it possible to assess the specific contributions of IL-1 to the acute phase changes induced by complex mixtures of cytokines. We utilized IL-1ra to define the contribution of IL-1 to the effects of conditioned medium from LPS-stimulated monocytes on production of the positive acute phase proteins C-reactive protein, serum amyloid A, fibrinogen, alpha 1-protease inhibitor, complement component C3, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, and ceruloplasmin and the negative acute phase proteins albumin and transferrin in Hep 3B cells. Induction of C-reactive protein and serum amyloid A was essentially abolished, induction of complement component C3 and alpha 1-acid glycoprotein was moderately decreased and induction of fibrinogen was enhanced. In contrast, there was no significant effect of IL-1ra on induction by conditioned medium of alpha 1-protease inhibitor, alpha 1-antichymotrypsin, or ceruloplasmin. IL-1ra partially blocked the down-regulatory effects of conditioned medium on both of the negative acute phase proteins we studied--albumin and transferrin. These findings enhance our understanding of the contribution of IL-1 to the acute phase response. In addition, they indicate that IL-1ra in vivo may influence synthesis of both positive and negative acute phase proteins. PMID:7682588

  20. Classification epitopes in groups based on their protein family

    PubMed Central

    2015-01-01

    Background The humoral immune system response is based on the interaction between antibodies and antigens for the clearance of pathogens and foreign molecules. The interaction between these proteins occurs at specific positions known as antigenic determinants or B-cell epitopes. The experimental identification of epitopes is costly and time consuming. Therefore the use of in silico methods, to help discover new epitopes, is an appealing alternative due the importance of biomedical applications such as vaccine design, disease diagnostic, anti-venoms and immune-therapeutics. However, the performance of predictions is not optimal been around 70% of accuracy. Further research could increase our understanding of the biochemical and structural properties that characterize a B-cell epitope. Results We investigated the possibility of linear epitopes from the same protein family to share common properties. This hypothesis led us to analyze physico-chemical (PCP) and predicted secondary structure (PSS) features of a curated dataset of epitope sequences available in the literature belonging to two different groups of antigens (metalloproteinases and neurotoxins). We discovered statistically significant parameters with data mining techniques which allow us to distinguish neurotoxin from metalloproteinase and these two from random sequences. After a five cross fold validation we found that PCP based models obtained area under the curve values (AUC) and accuracy above 0.9 for regression, decision tree and support vector machine. Conclusions We demonstrated that antigen's family can be inferred from properties within a single group of linear epitopes (metalloproteinases or neurotoxins). Also we discovered the characteristics that represent these two epitope groups including their similarities and differences with random peptides and their respective amino acid sequence. These findings open new perspectives to improve epitope prediction by considering the specific antigen

  1. Performance of Protein Induced by Vitamin K Absence or Antagonist-II (PIVKA-II) for Hepatocellular Carcinoma Screening in Chinese Population

    PubMed Central

    Yu, Rentao; Ding, Shitao; Tan, Wenting; Tan, Shun; Tan, Zhaoxia; Xiang, Shiqing; Zhou, Yi; Mao, Qing; Deng, Guohong

    2015-01-01

    Background: Alpha-fetoprotein (AFP) has long been used as an effective biomarker for hepatocellular carcinoma (HCC) screening; however, not all HCC patients can be detected with an elevated AFP level, especially in early HCC patients. Protein Induced by vitamin K absence or antagonist-II (PIVKA-II) is another serum biomarker linked to HCC; however, sensitivity and specificity remain controversial and data in Chinese groups is even rarer. Objectives: To evaluate the performance of PIVKA-II alone and combined with AFP in HCC screening in Chinese population. Patients and Methods: This retrospective study enrolled 150 HCC patients in Southwest Hospital, of which 16 patients were excluded due to lack of basic information. A total of 347 patients with hepatitis B, 105 with non-HCC cancers and 53 healthy people were enrolled as controls. Levels of AFP and PIVKA-II were measured by chemiluminescence enzyme immunoassay (CLEIA) and chemiluminescent microparticle Immunoassay (CMIA), respectively. Results: The sensitivity and specificity of PIVKA-II were 74.6% and 67.8% at a cutoff of 40 mAU/mL and 64.2% and 89.7% at a cutoff of 200 mAU/mL. The sensitivity and specificity of AFP were 76.7% and 65.0% at a cutoff of 20 ng/mL and 60.4% and 88.9% at a cutoff of 195.23 ng/mL. The combination of two markers had a sensitivity and specificity of 91.1% and 41.0%, respectively. The area under the receiving operating curve (AUROC) for PIVKA-II (0.756, 95% confidence interval, CI: 0.695 - 0.816) was less than the AUROC for AFP (0.823, 95% CI: 0.780 - 0.865), and in combination, the AUROC increased to 0.843 (95% CI: 0.801 - 0.885). Conclusions: PIVKA-II was as efficient as AFP when used as a single marker for HCC screening and the combination of two biomarkers gave a better performance. PMID:26300931

  2. The protective M proteins of the equine group C streptococci.

    PubMed

    Timoney, J F; Mukhtar, M M

    1993-11-01

    The group C streptococci are the most commonly isolated bacteria from disease states in the horse. Important virulence factors of S. equi and S. zooepidemicus are the hyaluronic acid capsule and the antiphagocytic fibrillar M protein located on the surface of the cell wall and extending into and through the capsule. The hyaluronic acid capsule is non-antigenic and so is not involved in protective immunity. The M protein, a superantigen, elicits very strong B and T cell responses that may result in protective immunity mediated by opsonic antibodies in plasma and by locally synthesized IgG and IgA on the pharyngeal mucosa. However, vaccines based on acid or mutanolysin extracted M protein do not confer a high level of protection against field exposure. Protective antibodies to S. equi or S. zooepidemicus can in part be assayed by the bactericidal test that measures opsonization for equine neutrophils. A mouse-challenge model has also been used to test immunizing potency of streptococcal extracts and in a passive protection test for protective antibody. There is as yet no means of distinguishing protective opsonic or mucosal antibodies from other antibodies produced against the many epitopes on the M molecule. PMID:8116194

  3. The angiotensin II receptor antagonist, losartan, enhances regulator of G protein signaling 2 mRNA expression in vascular smooth muscle cells of Wistar rats.

    PubMed

    Wu, Yaqiong; Nakagawa, Suguru; Takahashi, Hidenori; Kawabata, Yukari; Suzuki, Etsu; Uehara, Yoshio

    2016-05-01

    Angiotensin II (Ang II) reportedly enhances regulator of G-protein signaling 2 (RGS2), thus making a negative feedback loop for Ang II signal transduction. However, few studies have reported whether Ang II receptor (ATR) antagonists influence RGS2 mRNA expression. We investigated RGS2 mRNA expression when Ang II binding to ATR was blocked with Ang II subtype-1 receptor (AT1R) blockers using vascular smooth muscle cells from the thoracic aorta of male Wistar rats. RGS2 mRNA expression significantly increased with Ang II stimulation, and this increase was almost completely abolished by olmesartan, a potent AT1R-specific blocker. Ang II subtype-2 receptor (AT2R) was not involved in Ang II-mediated RGS expression. In contrast, the AT1R blocker, losartan, partially decreased Ang II-mediated RGS2 mRNA expression because this antagonist directly stimulated RGS2 mRNA expression in Ang II-free medium. EXP3174, which is an active metabolite of losartan, almost completely blunted Ang II-mediated RGS2 mRNA expression without direct stimulation of RGS2 mRNA expression. Moreover, pretreatment with olmesartan abolished Ang II-mediated RGS2 mRNA expression. Treatment with a protein kinase C inhibitor partially decreased losartan-mediated RGS2 mRNA expression. These results suggest that AT1R blockers inhibit RGS2 mRNA expression in response to Ang II via an AT1R-mediated mechanism. However, the AT1R blocker, losartan, behaves as a direct agonist for RGS2 mRNA expression via AT1R through protein kinase C-dependent and -independent pathways. In conclusion, losartan exhibits dual effects on RGS2 mRNA expression, and the direct upregulation of RGS2 mRNA expression may provide a new strategy for the treatment of hypertension. PMID:26763849

  4. Effects of tiflucarbine as a dual protein kinase C/calmodulin antagonist on proliferation of human keratinocytes and release of reactive oxygen species from human leukocytes.

    PubMed

    Hegemann, L; Fruchtmann, R; Bonnekoh, B; Schmidt, B H; Traber, J; Mahrle, G; Müller-Peddinghaus, R; van Rooijen, L A

    1991-01-01

    Various studies have suggested that calmodulin (CaM) is involved in the pathophysiology of psoriasis. Protein kinase C (PKC) is also accepted as playing a regulatory role in cell proliferation as well as in inflammatory processes. Therefore, we investigated the effects of the known CaM antagonist tiflucarbine (BAY/TVX P 4495) on two cellular systems related to the major clinical symptoms of psoriasis: proliferation of cultured human keratinocytes (HaCa T cell line) and release of reactive oxygen species (ROS) from human polymorphonuclear leukocytes (PMNL). Tiflucarbine inhibited both cellular responses in a dose dependent manner. Furthermore, tiflucarbine directly affected PKC, and may thus be considered to be a dual PKC/CaM antagonist with putative antipsoriatic activity. The effects of tiflucarbine on the different parameters were compared with those of the structurally unrelated dual PKC/CaM inhibitor W-7 and those of the potent PKC inhibitor staurosporine. The potencies of all three compounds were found to be in the same range as their PKC-inhibiting potency. Our data indicate that PKC, rather than CaM, may play a regulatory role in the release of ROS as well as in keratinocyte proliferation. Therefore, inhibition of PKC in general might have a therapeutic benefit in psoriasis. PMID:1801655

  5. The correlation between follicular fluid pregnancy-associated plasma protein A levels, fertilization, and embryo quality in GnRH agonist and GnRH antagonist protocols in ART cycles

    PubMed Central

    Dehghani Firouzabadi, Razieh; Mohammadian, Farnaz; Mashayekhy, Mehri; Davar, Robab; Eftekhar, Maryam

    2012-01-01

    Background: Determination of oocyte fertilization and embryo quality are one of the most important purposes in ART cycles. Follicular fluid provides an important microenvironment for development of oocytes and some biochemical characteristics of the follicular fluid, such as pregnancy-associated plasma protein-A (PAPP-A), may play an important role in prediction of success rate of ART. Objective: This study was performed to evaluate whether there was any difference in follicular fluid PAPP-A, fertilization, and embryo quality between GnRH agonist long protocol and flexible GnRH antagonist multiple-dose protocol in ART cycles. Materials and Methods: A total of 100 women who were candidates for ART were enrolled the study and were divided into two groups, GnRH agonist (GnRHa) long protocol (n=51) and flexible GnRH antagonist (GnRHant) multiple-dose protocol (n=49). Follicular fluid sample was obtained from a single mature follicle and follicular fluid PAPP-A level, fertilization and embryo quality of the same oocyte were evaluated in both groups. Results: There was no significant difference in the mean levels of follicular fluid PAPP-A between the GnRHa protocol and GnRHant protocol (3.5±1.4 vs. 3.8±1.9, respectively). The mean levels of follicular fluid PAPP-A in fertilized oocyte and good quality embryo were comparable in GnRHa and GnRHant protocols. Conclusion: Our data indicated that no differences of follicular fluid PAPP-A levels were observed between cycles using GnRHa long protocol and those of using flexible GnRHant multiple-dose protocol. PMID:25246915

  6. Bisphenol A binds to Ras proteins and competes with guanine nucleotide exchange: implications for GTPase-selective antagonists.

    PubMed

    Schöpel, Miriam; Jockers, Katharina F G; Düppe, Peter M; Autzen, Jasmin; Potheraveedu, Veena N; Ince, Semra; Yip, King Tuo; Heumann, Rolf; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

    2013-12-12

    We show for the first time that bisphenol A (10) has the capacity to interact directly with K-Ras and that Rheb weakly binds to bisphenol A (10) and 4,4'-biphenol derivatives. We have characterized these interactions at atomic resolution suggesting that these compounds sterically interfere with the Sos-mediated nucleotide exchange in H- and K-Ras. We show that 4,4'-biphenol (5) selectively inhibits Rheb signaling and induces cell death suggesting that this compound might be a novel candidate for treatment of tuberous sclerosis-mediated tumor growth. Our results propose a new mode of action for bisphenol A (10) that advocates a reduced exposure to this compound in our environment. Our data may lay the foundation for the future design of GTPase-selective antagonists with higher affinity to benefit of the treatment of cancer because K-Ras inhibition is regarded to be a promising strategy with a potential therapeutic window for targeting Sos in Ras-driven tumors. PMID:24266771

  7. A phase I/IIa clinical trial of a recombinant Rho protein antagonist in acute spinal cord injury.

    PubMed

    Fehlings, Michael G; Theodore, Nicholas; Harrop, James; Maurais, Gilles; Kuntz, Charles; Shaffrey, Chris I; Kwon, Brian K; Chapman, Jens; Yee, Albert; Tighe, Allyson; McKerracher, Lisa

    2011-05-01

    Multiple lines of evidence have validated the Rho pathway as important in controlling the neuronal response to growth inhibitory proteins after central nervous system (CNS) injury. A drug called BA-210 (trademarked as Cethrin(®)) blocks activation of Rho and has shown promise in pre-clinical animal studies in being used to treat spinal cord injury (SCI). This is a report of a Phase I/IIa clinical study designed to test the safety and tolerability of the drug, and the neurological status of patients following the administration of a single dose of BA-210 applied during surgery following acute SCI. Patients with thoracic (T2-T12) or cervical (C4-T1) SCI were sequentially recruited for this dose-ranging (0.3 mg to 9 mg Cethrin), multi-center study of 48 patients with complete American Spinal Injury Association assessment (ASIA) A. Vital signs; clinical laboratory tests; computed tomography (CT) scans of the spine, head, and abdomen; magnetic resonance imaging (MRI) of the spine, and ASIA assessment were performed in the pre-study period and in follow-up periods out to 1 year after treatment. The treatment-emergent adverse events that were reported were typical for a population of acute SCI patients, and no serious adverse events were attributed to the drug. The pharmacokinetic analysis showed low levels of systemic exposure to the drug, and there was high inter-patient variability. Changes in ASIA motor scores from baseline were low across all dose groups in thoracic patients (1.8±5.1) and larger in cervical patients (18.6±19.3). The largest change in motor score was observed in the cervical patients treated with 3 mg of Cethrin in whom a 27.3±13.3 point improvement in ASIA motor score at 12 months was observed. Approximately 6% of thoracic patients converted from ASIA A to ASIA C or D compared to 31% of cervical patients and 66% for the 3-mg cervical cohort. Although the patient numbers are small, the observed motor recovery in this open-label trial

  8. Cooperative and Antagonistic Contributions of Two Heterochromatin Proteins to Transcriptional Regulation of the Drosophila Sex Determination Decision

    PubMed Central

    Li, Hui; Rodriguez, Janel; Yoo, Youngdong; Shareef, Momin Mohammed; Badugu, RamaKrishna

    2011-01-01

    Eukaryotic nuclei contain regions of differentially staining chromatin (heterochromatin), which remain condensed throughout the cell cycle and are largely transcriptionally silent. RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability. Here we report a similar phenotype for the telomeric partner of HP1, HOAP, and roles for both proteins in regulating the Drosophila sex determination pathway. Specifically, these proteins regulate the critical decision in this pathway, firing of the establishment promoter of the masterswitch gene, Sex-lethal (Sxl). Female-specific activation of this promoter, SxlPe, is essential to females, as it provides SXL protein to initiate the productive female-specific splicing of later Sxl transcripts, which are transcribed from the maintenance promoter (SxlPm) in both sexes. HOAP mutants show inappropriate SxlPe firing in males and the concomitant inappropriate splicing of SxlPm-derived transcripts, while females show premature firing of SxlPe. HP1 mutants, by contrast, display SxlPm splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with SxlPe sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to SxlPe are severely compromised. Our genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at SxlPe. PMID:21695246

  9. Using the MCoTI-II Cyclotide Scaffold To Design a Stable Cyclic Peptide Antagonist of SET, a Protein Overexpressed in Human Cancer.

    PubMed

    D'Souza, Charlotte; Henriques, Sónia Troeira; Wang, Conan K; Cheneval, Olivier; Chan, Lai Yue; Bokil, Nilesh J; Sweet, Matthew J; Craik, David J

    2016-01-19

    The SET protein is a promising drug target in cancer therapy, because of its ability to inhibit the function of the tumor suppressor gene protein phosphatase 2A (PP2A). COG peptides, derived from apolipoprotein E (apoE), are potent antagonists of SET; they induce cytotoxicity in cancer cells upon binding to intracellular SET and modulate the nuclear factor kappa B (NF-κB) signaling pathway. However, the therapeutic potential of COG peptides is limited, because of their poor proteolytic stability and low bioavailability. In this study, the COG peptide, COG1410, was stabilized by grafting it onto the ultrastable cyclic peptide scaffold, Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The grafted MCoTI-II peptides were cytotoxic to a cancer cell line and showed high stability in human serum. The most potent grafted MCoTI-II peptide inhibited lipopolysaccharide (LPS)-mediated activation of NF-κB in murine macrophages. Overall, this study demonstrates the application of the MCoTI-II scaffold for the development of stable peptide drugs for cancer therapy. PMID:26685975

  10. P2X7R large pore is partially blocked by pore forming proteins antagonists in astrocytes.

    PubMed

    Faria, Robson X; Reis, Ricardo A M; Ferreira, Leonardo G B; Cezar-de-Mello, Paula F T; Moraes, Milton O

    2016-06-01

    The ATP-gated P2X7R (P2X7R) is a channel, which is involved in events, such as inflammation, cell death, and pain. The most intriguing event concerning P2X7R functions is the phenomenon of pore dilation. Once P2X7R is activated, the permeability of the plasma membrane becomes higher, leading to the permeation of 1000 Da-weight solutes. The mechanisms involved in this process remain unclear. Nevertheless, this event is not exclusively through P2X7R, as other proteins may form large pores in the plasma membrane. Recent evidence concerning pore formation reveals putative P2X7R and other pores-associated protein complexes, revealing cross-interactive pharmacological and biophysical issues. In this work, we showed results that corroborated with cross-interactive aspects with P2X7R and pores in astrocytes. These cells expressed most of the pores, including P2X7R. We discovered that different pore types open with peculiar characteristics, as both anionic and cationic charged solutes permeate the plasma membrane, following P2X7R activation. Moreover, we showed that both synergic and additive relationships are found within P2X7, cationic, and anionic large pores. Therefore, our data suggest that other protein-related pores are assembled following the formation of P2X7R pore. PMID:26830892

  11. Periparturient cortisol, acute phase cytokine, and acute phase protein profiles of gilts housed in groups or stalls during gestation.

    PubMed

    Sorrells, A D; Eicher, S D; Harris, M J; Pajor, E A; Richert, B T

    2007-07-01

    Use of gestation stalls in pork production remains a controversial topic in animal welfare. Immune status and measures are frequently used to assess stress levels and thus well-being of confined animals. The important welfare issue of close confinement among gestating gilts was tested by quantifying cortisol, acute phase cytokine, and acute phase protein pro-files before and after farrowing of gilts housed in 2 systems. Landrace x Yorkshire crossbred gilts housed in groups of 4 (group, n = 8) in pens (3.9 x 2.4 m with 4 individual feeding spaces, 9.36 m(2) total or 2.34 m(2)/gilt) were compared with gilts housed in standard industry stalls (stall, n = 16; 2.2 x 0.6 m, 1.32 m(2)/gilt). Floors were fully slatted, and a substrate was not provided for either system. Cortisol was determined from saliva on d 105 of gestation, 1 h after moving the gilts into farrowing stalls (d 111), and 24 h and 7 d after farrowing. Cortisol was greater (P = 0.04) for group gilts compared with stall gilts 1 h after moving them into farrowing stalls and 24 h after farrowing. Cortisol concentrations decreased (P = 0.001) over time. Leukocyte mRNA expression of IL-1, IL-1 receptor antagonist, and tumor necrosis factor-alpha was determined by quantitative, reverse transcription PCR on d 35, 63, and 91 of gestation and 72 h after farrowing. Cytokine mRNA expression of peripheral blood mononuclear cells did not differ between housing systems for IL-1, its receptor antagonist, or for tumor necrosis factor-alpha. Acute phase proteins, including fibrinogen, haptoglobin, and alpha(1)-acid glycoprotein were determined for plasma samples taken at d 35, 63, and 91 of gestation and 72 h and 14 d after farrowing. In contrast to cortisol, plasma fibrinogen concentrations increased (P < 0.005) over time. Haptoglobin did not differ between treatments (P > 0.10). Stall gilts tended to have greater (P = 0.07) plasma alpha(1)-acid glycoprotein concentrations than group animals at d 35 of gestation and d 14

  12. Pepper protein phosphatase type 2C, CaADIP1 and its interacting partner CaRLP1 antagonistically regulate ABA signalling and drought response.

    PubMed

    Lim, Chae Woo; Lee, Sung Chul

    2016-07-01

    Abscisic acid (ABA) is a key phytohormone that regulates plant growth and developmental processes, including seed germination and stomatal closing. Here, we report the identification and functional characterization of a novel type 2C protein phosphatase, CaADIP1 (Capsicum annuum ABA and Drought-Induced Protein phosphatase 1). The expression of CaADIP1 was induced in pepper leaves by ABA, drought and NaCl treatments. Arabidopsis plants overexpressing CaADIP1 (CaADIP1-OX) exhibited an ABA-hyposensitive and drought-susceptible phenotype. We used a yeast two-hybrid screening assay to identify CaRLP1 (Capsicum annuum RCAR-Like Protein 1), which interacts with CaADIP1 in the cytoplasm and nucleus. In contrast to CaADIP1-OX plants, CaRLP1-OX plants displayed an ABA-hypersensitive and drought-tolerant phenotype, which was characterized by low levels of transpirational water loss and increased expression of stress-responsive genes relative to those of wild-type plants. In CaADIP1-OX/CaRLP1-OX double transgenic plants, ectopic expression of the CaRLP1 gene led to strong suppression of CaADIP1-induced ABA hyposensitivity during the germinative and post-germinative stages, indicating that CaADIP1 and CaRLP1 act in the same signalling pathway and CaADIP1 functions downstream of CaRLP1. Our results indicate that CaADIP1 and its interacting partner CaRLP1 antagonistically regulate the ABA-dependent defense signalling response to drought stress. PMID:26825039

  13. The G protein-coupled receptor kinase-2 is a TGFbeta-inducible antagonist of TGFbeta signal transduction.

    PubMed

    Ho, Joanne; Cocolakis, Eftihia; Dumas, Victor M; Posner, Barry I; Laporte, Stéphane A; Lebrun, Jean-Jacques

    2005-09-21

    Signaling from the activin/transforming growth factor beta (TGFbeta) family of cytokines is a tightly regulated process. Disregulation of TGFbeta signaling is often the underlying basis for various cancers, tumor metastasis, inflammatory and autoimmune diseases. In this study, we identify the protein G-coupled receptor kinase 2 (GRK2), a kinase involved in the desensitization of G protein-coupled receptors (GPCR), as a downstream target and regulator of the TGFbeta-signaling cascade. TGFbeta-induced expression of GRK2 acts in a negative feedback loop to control TGFbeta biological responses. Upon TGFbeta stimulation, GRK2 associates with the receptor-regulated Smads (R-Smads) through their MH1 and MH2 domains and phosphorylates their linker region. GRK2 phosphorylation of the R-Smads inhibits their carboxyl-terminal, activating phosphorylation by the type I receptor kinase, thus preventing nuclear translocation of the Smad complex, leading to the inhibition of TGFbeta-mediated target gene expression, cell growth inhibition and apoptosis. Furthermore, we demonstrate that GRK2 antagonizes TGFbeta-induced target gene expression and apoptosis ex vivo in primary hepatocytes, establishing a new role for GRK2 in modulating single-transmembrane serine/threonine kinase receptor-mediated signal transduction. PMID:16121194

  14. The interferon signaling antagonist function of yellow fever virus NS5 protein is activated by Type I interferon

    PubMed Central

    Rajsbaum, Ricardo; Macleod, Jesica M. Levingston; Pisanelli, Giuseppe; Pham, Alissa; Ayllon, Juan; Miorin, Lisa; Martinez, Carles; tenOever, Benjamin R; García-Sastre, Adolfo

    2014-01-01

    Summary To successfully establish infection Flaviviruses have to overcome the antiviral state induced by type I interferon (IFN-I). The nonstructural NS5 proteins of several flaviviruses antagonize IFN-I signaling. Here we show that yellow fever virus (YFV) inhibits IFN-I signaling through a unique mechanism that involves binding of YFV NS5 to the IFN-activated transcription factor STAT2 only in cells that have been stimulated with IFN-I. This NS5-STAT2 interaction requires IFN-I-induced tyrosine phosphorylation of STAT1 and the K63-linked polyubiquitination at a lysine in the N-terminal region of YFV NS5. We identified TRIM23 as the E3 ligase that interacts with and polyubiquitinates YFV NS5 to promote its binding to STAT2 and trigger IFN-I signaling inhibition. Our results demonstrate the importance of YFV NS5 in overcoming the antiviral action of IFN-I and offer a unique example of a viral protein that is activated by the same host pathway that it inhibits. PMID:25211074

  15. Follistatin-like 1 (Fstl1) is a bone morphogenetic protein (BMP) 4 signaling antagonist in controlling mouse lung development.

    PubMed

    Geng, Yan; Dong, Yingying; Yu, Mingyan; Zhang, Long; Yan, Xiaohua; Sun, Jingxia; Qiao, Long; Geng, Huixia; Nakajima, Masahiro; Furuichi, Tatsuya; Ikegawa, Shiro; Gao, Xiang; Chen, Ye-Guang; Jiang, Dianhua; Ning, Wen

    2011-04-26

    Lung morphogenesis is a well orchestrated, tightly regulated process through several molecular pathways, including TGF-β/bone morphogenetic protein (BMP) signaling. Alteration of these signaling pathways leads to lung malformation. We investigated the role of Follistatin-like 1 (Fstl1), a secreted follistatin-module-containing glycoprotein, in lung development. Deletion of Fstl1 in mice led to postnatal lethality as a result of respiratory failure. Analysis of the mutant phenotype showed that Fstl1 is essential for tracheal cartilage formation and alveolar maturation. Deletion of the Fstl1 gene resulted in malformed tracheal rings manifested as discontinued rings and reduced ring number. Fstl1-deficient mice displayed septal hypercellularity and end-expiratory atelectasis, which were associated with impaired differentiation of distal alveolar epithelial cells and insufficient production of mature surfactant proteins. Mechanistically, Fstl1 interacted directly with BMP4, negatively regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced surfactant gene expression. Reducing BMP signaling activity by Noggin rescued pulmonary atelectasis of Fstl1-deficient mice. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in part, through BMP4 signaling. PMID:21482757

  16. Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2.

    PubMed

    Roy, B C; Subramaniam, D; Ahmed, I; Jala, V R; Hester, C M; Greiner, K A; Haribabu, B; Anant, S; Umar, S

    2015-08-20

    The enhancer of zeste homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/β-catenin signaling. We aimed at investigating EZH2's role in epigenetically regulating Wnt/β-catenin signaling following bacterial infection. NIH:Swiss outbred and Apc(Min/+) mice were infected with CR (10(8) CFU); BLT1(-/-)Apc(Min/+) mice, azoxymethane (AOM)/dextran sodium sulfate (DSS)-treated mice and de-identified human adenocarcinoma samples were the models of colon cancer. Following infection with wild-type but not mutant CR, elevated EZH2 levels in the crypt at days 6 and 12 (peak hyperplasia) coincided with increases in H3K27me3 and β-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3's occupancy on WIF1 (Wnt inhibitory factor 1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via small interfering RNA or EZH2-inhibitor deazaneplanocin A (Dznep) either alone or in combination with histone deacetylase inhibitor suberoylanilide hydroxamic acid, WIF1 promoter activity increased significantly while the overexpression of EZH2 attenuated WIF1 reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels, whereas H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, although β-catenin levels were lower in EZH2-knocked down cells, F681Y mutants exhibited only partial reduction in β-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days 6 and 12 post infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and

  17. Prolonged Survival in a Case of Chemotherapy-Sensitive Gastric Cancer That Produced Alpha-Fetoprotein and Protein Induced by Vitamin K Antagonist-II.

    PubMed

    Ogasawara, Naotaka; Takahashi, Emiko; Matsumoto, Tomoko; Amaike, Manami; Nohara, Mako; Nagao, Kazuhiro; Ebi, Masahide; Funaki, Yasushi; Sasaki, Makoto; Kasugai, Kunio

    2015-01-01

    The number of reported cases of alpha-fetoprotein (AFP)-producing gastric cancer has gradually increased, with a reported prevalence of 1.3-1.5% of all gastric cancer cases. However, reports of gastric cancer accompanied by elevated serum levels of both AFP and protein induced by vitamin K antagonist-II (PIVKA-II) are rare. The prognosis of AFP- and PIVKA-II-producing gastric cancer has been reported to be very poor because the tumor cells were considered to have a high malignant potential and the cancer progressed rapidly. We described a case of gastric cancer producing AFP and PIVKA-II in which chemotherapy was effective and resulted in prolonged survival, and these two tumor markers were useful for monitoring the treatment response. Routine health screening using upper abdominal ultrasonography revealed hepatic tumors in an apparently healthy 65-year-old man. Whole-body computed tomography (CT) revealed multiple hepatic tumors, and an esophagogastroduodenoscopy (EGD) revealed a Bormann type 3 tumor in the lower stomach. A biopsy specimen confirmed that the tumor was immunohistochemically positive for AFP, PIVKA-II, and human epidermal growth factor receptor 2. After chemotherapy, the gastric tumor appeared as a small elevated lesion on EGD, and CT revealed a remarkable reduction in the size of the metastatic liver tumors. The patient is still alive, 35 months after the initial chemotherapy. PMID:26034473

  18. The F-box-containing protein UFO and AGAMOUS participate in antagonistic pathways governing early petal development in Arabidopsis

    PubMed Central

    Durfee, Tim; Roe, Judith L.; Sessions, R. Allen; Inouye, Carla; Serikawa, Kyle; Feldmann, Kenneth A.; Weigel, Detlef; Zambryski, Patricia C.

    2003-01-01

    The UNUSUAL FLORAL ORGANS (UFO) gene is required for multiple processes in the developing Arabidopsis flower, including the proper patterning and identity of both petals and stamens. The gene encodes an F-box-containing protein, UFO, which interacts physically and genetically with the Skp1 homolog, ASK1. In this report, we describe four ufo alleles characterized by the absence of petals, which uncover another role for UFO in promoting second whorl development. This UFO-dependent pathway is required regardless of the second whorl organ to be formed, arguing that it affects a basic process acting in parallel with those establishing organ identity. However, the pathway is dispensable in the absence of AGAMOUS (AG), a known inhibitor of petal development. In situ hybridization results argue that AG is not transcribed in the petal region, suggesting that it acts non-cell-autonomously to inhibit second whorl development in ufo mutants. These results are combined into a genetic model explaining early second whorl initiation/proliferation, in which UFO functions to inhibit an AG-dependent activity. PMID:12826617

  19. The F-box-containing protein UFO and AGAMOUS participate in antagonistic pathways governing early petal development in Arabidopsis.

    PubMed

    Durfee, Tim; Roe, Judith L; Sessions, R Allen; Inouye, Carla; Serikawa, Kyle; Feldmann, Kenneth A; Weigel, Detlef; Zambryski, Patricia C

    2003-07-01

    The UNUSUAL FLORAL ORGANS (UFO) gene is required for multiple processes in the developing Arabidopsis flower, including the proper patterning and identity of both petals and stamens. The gene encodes an F-box-containing protein, UFO, which interacts physically and genetically with the Skp1 homolog, ASK1. In this report, we describe four ufo alleles characterized by the absence of petals, which uncover another role for UFO in promoting second whorl development. This UFO-dependent pathway is required regardless of the second whorl organ to be formed, arguing that it affects a basic process acting in parallel with those establishing organ identity. However, the pathway is dispensable in the absence of AGAMOUS (AG), a known inhibitor of petal development. In situ hybridization results argue that AG is not transcribed in the petal region, suggesting that it acts non-cell-autonomously to inhibit second whorl development in ufo mutants. These results are combined into a genetic model explaining early second whorl initiation/proliferation, in which UFO functions to inhibit an AG-dependent activity. PMID:12826617

  20. Selective orexin receptor antagonists.

    PubMed

    Lebold, Terry P; Bonaventure, Pascal; Shireman, Brock T

    2013-09-01

    The orexin, or hypocretin, neuropeptides (orexin-A and orexin-B) are produced on neurons in the hypothalamus which project to key areas of the brain that control sleep-wake states, modulation of food intake, panic, anxiety, emotion, reward and addictive behaviors. These neuropeptides exert their effects on a pair of G-protein coupled receptors termed the orexin-1 (OX1) and orexin-2 (OX2) receptors. Emerging biology suggests the involvement of these receptors in psychiatric disorders as they are thought to play a key role in the regulation of multiple systems. This review is intended to highlight key selective OX1 or OX2 small-molecule antagonists. PMID:23891187

  1. Group 1 LEA proteins, an ancestral plant protein group, are also present in other eukaryotes, and in the archeae and bacteria domains.

    PubMed

    Campos, F; Cuevas-Velazquez, C; Fares, M A; Reyes, J L; Covarrubias, A A

    2013-10-01

    Water is an essential element for living organisms, such that various responses have evolved to withstand water deficit in all living species. The study of these responses in plants has had particular relevance given the negative impact of water scarcity on agriculture. Among the molecules highly associated with plant responses to water limitation are the so-called late embryogenesis abundant (LEA) proteins. These proteins are ubiquitous in the plant kingdom and accumulate during the late phase of embryogenesis and in vegetative tissues in response to water deficit. To know about the evolution of these proteins, we have studied the distribution of group 1 LEA proteins, a set that has also been found beyond the plant kingdom, in Bacillus subtilis and Artemia franciscana. Here, we report the presence of group 1 LEA proteins in green algae (Chlorophyita and Streptophyta), suggesting that these group of proteins emerged before plant land colonization. By sequence analysis of public genomic databases, we also show that 34 prokaryote genomes encode group 1 LEA-like proteins; two of them belong to Archaea domain and 32 to bacterial phyla. Most of these microbes live in soil-associated habitats suggesting horizontal transfer from plants to bacteria; however, our phylogenetic analysis points to convergent evolution. Furthermore, we present data showing that bacterial group 1 LEA proteins are able to prevent enzyme inactivation upon freeze-thaw treatments in vitro, suggesting that they have analogous functions to plant LEA proteins. Overall, data in this work indicate that LEA1 proteins' properties might be relevant to cope with water deficit in different organisms. PMID:23861025

  2. Expression of group B protective surface protein (BPS) by invasive and colonizing isolates of group B streptococci.

    PubMed

    Flores, Aurea E; Chhatwal, G S; Hillier, Sharon L; Baker, Carol J; Ferrieri, Patricia

    2014-12-01

    Group B protective surface protein (BPS) is expressed on the cell surface of some group B streptococcal (GBS) (Streptococcus agalactiae) strains and adds to the identification by capsular polysaccharide (CPS), and c or R proteins. We investigated the prevalence of BPS among GBS clinical isolates (303 invasive, 4122 colonizing) collected over 11 years in four American cities. Hot HCl cell extracts were tested by immunoprecipitation in agarose with rabbit antisera to BPS; the alpha (α) and beta (β) components of c protein; R1, R3, and R4 species of R protein; and CPS serotypes Ia-VIII. BPS was found in 155 isolates (seven invasive, 148 colonizing). Of these, 87 were Ia, 37 II, 20 V; none were III. BPS was expressed usually with another protein: a species of R by 87 or a component of c by 39. The predominant CPS/protein profiles with BPS were Ia/R1,BPS and II/c(α + β),BPS. Thus, along with CPS serotype and other surface proteins, BPS can be a valuable marker for precise strain characterization of unique GBS clinical isolates with complex surface protein profiles. PMID:25108378

  3. Analysis of spiroplasma proteins: contribution to the taxonomy of group IV spiroplasmas and the characterization of spiroplasma protein antigens.

    PubMed Central

    Mouches, C.; Candresse, T.; McGarrity, G. J.; Bové, J. M.

    1983-01-01

    Spiroplasma strains of group IV were compared by two-dimensional protein analyses on polyacrylamide gels. Although considerable diversity was evident, the assemblages studied were less heterogeneous than the known strains of group I. Two electrophoretic techniques were used to identify spiroplasma proteins that had been used to immunize rabbits. These included monoclonal antibodies prepared against Spiroplasma citri. In the first technique, protein antigens were purified by immunoaffinity chromatography, then identified with SDS-PAGE. In the second technique, spiroplasma proteins were first separated by SDS-PAGE, then antigens were identified by antibody binding to blot-transferred proteins. Finally, two-dimensional protein electrophoresis has been used as a source of immunogens to characterize monospecific antibodies against individual S. citri proteins. Images FIG. 1 FIG. 2 PMID:6206657

  4. [Genetic linkage of blood group, egg and serum protein and plumage color loci in chickens].

    PubMed

    Gintovt, V E; Novik, I E; Moiseeva, I G; Tolokoniikova, E V

    1976-01-01

    Genetic relationship of six blood group (A, B, C, D, E, x5), three egg (G2, G3, Ov) and one serum (Alb) protein loci and two plumage colour (I-dominant white, E-extended black) loci were investigated. 3250 gametes have been analysed for 21 loci combinations, 11 from them have never been studied on linkage. Blood group loci A, B, C, D, E, x5 segregated independently on egg protein loci G2, G3, and Ov, serum protein locus Alb and plumage colour locus E. No linkage was observed between blood group locus B and dominant white locus I. Close linkage for two egg protein loci G3 and Ov is confirmed. Independent segregation of investigated blood group, egg and serum protein loci suggests their localization on different autosomes in the chicken genome. The recent literature and the authors' data on genetic relationship between blood group, polymorphic protein loci and morphological traits are reviewed. PMID:1010323

  5. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

    PubMed Central

    Kudryavtsev, Denis; Bychkov, Maxim L.; Kulbatskii, Dmitrii S.; Kasheverov, Igor E.; Astapova, Maria V.; Feofanov, Alexey V.; Thomsen, Morten S.; Mikkelsen, Jens D.; Shenkarev, Zakhar O.; Tsetlin, Victor I.; Dolgikh, Dmitry A.; Kirpichnikov, Mikhail P.

    2016-01-01

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,—non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that

  6. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor.

    PubMed

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kudryavtsev, Denis; Bychkov, Maxim L; Kulbatskii, Dmitrii S; Kasheverov, Igor E; Astapova, Maria V; Feofanov, Alexey V; Thomsen, Morten S; Mikkelsen, Jens D; Shenkarev, Zakhar O; Tsetlin, Victor I; Dolgikh, Dmitry A; Kirpichnikov, Mikhail P

    2016-01-01

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that

  7. Quantitative evaluation of interaction force between functional groups in protein and polymer brush surfaces.

    PubMed

    Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

    2014-03-18

    To understand interactions between polymer surfaces and different functional groups in proteins, interaction forces were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Various polymer brush surfaces were systematically prepared by surface-initiated atom transfer radical polymerization as well-defined model surfaces to understand protein adsorption behavior. The polymer brush layers consisted of phosphorylcholine groups (zwitterionic/hydrophilic), trimethylammonium groups (cationic/hydrophilic), sulfonate groups (anionic/hydrophilic), hydroxyl groups (nonionic/hydrophilic), and n-butyl groups (nonionic/hydrophobic) in their side chains. The interaction forces between these polymer brush surfaces and different functional groups (carboxyl groups, amino groups, and methyl groups, which are typical functional groups existing in proteins) were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Furthermore, the amount of adsorbed protein on the polymer brush surfaces was quantified by surface plasmon resonance using albumin with a negative net charge and lysozyme with a positive net charge under physiological conditions. The amount of proteins adsorbed on the polymer brush surfaces corresponded to the interaction forces generated between the functional groups on the cantilever and the polymer brush surfaces. The weakest interaction force and least amount of protein adsorbed were observed in the case of the polymer brush surface with phosphorylcholine groups in the side chain. On the other hand, positive and negative surfaces generated strong forces against the oppositely charged functional groups. In addition, they showed significant adsorption with albumin and lysozyme, respectively. These results indicated that the interaction force at the functional group level might be

  8. The Polycomb Group Protein EED Interacts with YY1, and Both Proteins Induce Neural Tissue in Xenopus Embryos

    PubMed Central

    Satijn, David P. E.; Hamer, Karien M.; den Blaauwen, Jan; Otte, Arie P.

    2001-01-01

    Polycomb group (PcG) proteins form multimeric protein complexes which are involved in the heritable stable repression of genes. Previously, we identified two distinct human PcG protein complexes. The EED-EZH protein complex contains the EED and EZH2 PcG proteins, and the HPC-HPH PcG complex contains the HPC, HPH, BMI1, and RING1 PcG proteins. Here we show that YY1, a homolog of the Drosophila PcG protein pleiohomeotic (Pho), interacts specificially with the human PcG protein EED but not with proteins of the HPC-HPH PcG complex. Since YY1 and Pho are DNA-binding proteins, the interaction between YY1 and EED provides a direct link between the chromatin-associated EED-EZH PcG complex and the DNA of target genes. To study the functional significance of the interaction, we expressed the Xenopus homologs of EED and YY1 in Xenopus embryos. Both Xeed and XYY1 induce an ectopic neural axis but do not induce mesodermal tissues. In contrast, members of the HPC-HPH PcG complex do not induce neural tissue. The exclusive, direct neuralizing activity of both the Xeed and XYY1 proteins underlines the significance of the interaction between the two proteins. Our data also indicate a role for chromatin-associated proteins, such as PcG proteins, in Xenopus neural induction. PMID:11158321

  9. A mini review of the high mobility group proteins of insects.

    PubMed

    Aleporou-Marinou, Vassiliki; Marinou, Haroula; Patargias, Theocharis

    2003-10-01

    High mobility group (HMG) proteins are an abundant class of chromosomal proteins facilitate assembly of higher order structures. The mammalian HMG proteins have been grouped into three distinct families on the basis of their characteristic functional sequence: the HMGB, the HMGN, and the HMGA family. The HMG proteins of Drosophila melanogaster and Chironomus tentans are the best characterized dipteran insect HMG proteins. Three abundant members of this group of nonhistone proteins were detected in those insects. Two of them belong to the HMGB family and one to the HMGA family. The possible relatedness of these proteins to the formation of higher order nucleoprotein structures and their possible role in the regulation of transcription is discussed. PMID:14974679

  10. Effects of depolarization and NMDA antagonists on the role survival of cerebellar granule cells: a pivotal role for protein kinase C isoforms.

    PubMed

    Lin, W W; Wang, C W; Chuang, D M

    1997-06-01

    Primary cultures of cerebellar granule cells (CGCs) grown in high-K+ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3-4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time-dependently increased by 3.2-fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform-specific antibodies revealed an increase in levels of PKC alpha, gamma, mu, lambda, and iota from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC epsilon and betaII, respectively, whereas no significant change was observed for betaI. The isoforms of delta, theta, eta, and zeta were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC iota and epsilon were decreased, those of PKC alpha, betaI, betaII, gamma, and lambda were unchanged, whereas that of PKC mu was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2-4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low-K+ cultures, levels of PKC alpha, mu, lambda, and iota, but not the others, were markedly decreased. The NMDA receptor antagonists MK-801 and 2-amino-5-phosphopentanoic acid markedly prevented the age-induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK-801 was concomitant with the increases in levels of PKC gamma, lambda, iota, and mu at 10 and 14 DIV

  11. Contribution of Charged Groups to the Enthalpic Stabilization of the Folded States of Globular Proteins

    PubMed Central

    Dadarlat, Voichita M.; Post, Carol Beth

    2016-01-01

    In this paper we use the results from all atom MD simulations of proteins and peptides to assess individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the “unfolded state”) and charged amino acids in globular proteins (the “folded state”). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO−) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while NH3+ groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously. PMID:18303881

  12. Reductions in log P improved protein binding and clearance predictions enabling the prospective design of cannabinoid receptor (CB1) antagonists with desired pharmacokinetic properties.

    PubMed

    Ellsworth, Bruce A; Sher, Philip M; Wu, Ximao; Wu, Gang; Sulsky, Richard B; Gu, Zhengxiang; Murugesan, Natesan; Zhu, Yeheng; Yu, Guixue; Sitkoff, Doree F; Carlson, Kenneth E; Kang, Liya; Yang, Yifan; Lee, Ning; Baska, Rose A; Keim, William J; Cullen, Mary Jane; Azzara, Anthony V; Zuvich, Eva; Thomas, Michael A; Rohrbach, Kenneth W; Devenny, James J; Godonis, Helen E; Harvey, Susan J; Murphy, Brian J; Everlof, Gerry G; Stetsko, Paul I; Gudmundsson, Olafur; Johnghar, Susan; Ranasinghe, Asoka; Behnia, Kamelia; Pelleymounter, Mary Ann; Ewing, William R

    2013-12-12

    Several strategies have been employed to reduce the long in vivo half-life of our lead CB1 antagonist, triazolopyridazinone 3, to differentiate the pharmacokinetic profile versus the lead clinical compounds. An in vitro and in vivo clearance data set revealed a lack of correlation; however, when compounds with <5% free fraction were excluded, a more predictable correlation was observed. Compounds with log P between 3 and 4 were likely to have significant free fraction, so we designed compounds in this range to give more predictable clearance values. This strategy produced compounds with desirable in vivo half-lives, ultimately leading to the discovery of compound 46. The progression of compound 46 was halted due to the contemporaneous marketing and clinical withdrawal of other centrally acting CB1 antagonists; however, the design strategy successfully delivered a potent CB1 antagonist with the desired pharmacokinetic properties and a clean off-target profile. PMID:24182233

  13. Sub-grouping and sub-functionalization of the RIFIN multi-copy protein family

    PubMed Central

    Joannin, Nicolas; Abhiman, Saraswathi; Sonnhammer, Erik L; Wahlgren, Mats

    2008-01-01

    Background Parasitic protozoans possess many multicopy gene families which have central roles in parasite survival and virulence. The number and variability of members of these gene families often make it difficult to predict possible functions of the encoded proteins. The families of extra-cellular proteins that are exposed to a host immune response have been driven via immune selection to become antigenically variant, and thereby avoid immune recognition while maintaining protein function to establish a chronic infection. Results We have combined phylogenetic and function shift analyses to study the evolution of the RIFIN proteins, which are antigenically variant and are encoded by the largest multicopy gene family in Plasmodium falciparum. We show that this family can be subdivided into two major groups that we named A- and B-RIFIN proteins. This suggested sub-grouping is supported by a recently published study that showed that, despite the presence of the Plasmodium export (PEXEL) motif in all RIFIN variants, proteins from each group have different cellular localizations during the intraerythrocytic life cycle of the parasite. In the present study we show that function shift analysis, a novel technique to predict functional divergence between sub-groups of a protein family, indicates that RIFINs have undergone neo- or sub-functionalization. Conclusion These results question the general trend of clustering large antigenically variant protein groups into homogenous families. Assigning functions to protein families requires their subdivision into meaningful groups such as we have shown for the RIFIN protein family. Using phylogenetic and function shift analysis methods, we identify new directions for the investigation of this broad and complex group of proteins. PMID:18197962

  14. Vagal modulation of high mobility group box-1 protein mediates electroacupuncture-induced cardioprotection in ischemia-reperfusion injury

    PubMed Central

    Zhang, Juan; Yong, Yue; Li, Xing; Hu, Yu; Wang, Jian; Wang, Yong-qiang; Song, Wei; Chen, Wen-ting; Xie, Jian; Chen, Xue-mei; Lv, Xin; Hou, Li-li; Wang, Ke; Zhou, Jia; Wang, Xiang-rui; Song, Jian-gang

    2015-01-01

    Excessive release of high mobility group box-1 (HMGB1) protein from ischemic cardiomyocytes activates inflammatory cascades and enhances myocardial injury after reperfusion. Here we report evidence that electroacupuncture of mice at Neiguan acupoints can inhibit the up-regulation of cardiac HMGB1 following myocardial ischemia and attenuate the associated inflammatory responses and myocardial injury during reperfusion. These benefits of electroacupuncture were partially reversed by administering recombinant HMGB1 to the mice, and further potentiated by administering anti-HMGB1 antibody. Electroacupuncture-induced inhibition of HMGB1 release was markedly reduced by unilateral vagotomy or administration of nicotinic receptor antagonist, but not by chemical sympathectomy. The cholinesterase inhibitor neostigmine mimicked the effects of electroacupuncture on HMGB1 release and myocardial ischemia reperfusion injury. Culture experiments with isolated neonatal cardiomyocytes showed that acetylcholine, but not noradrenaline, inhibited hypoxia-induced release of HMGB1 via a α7nAchR-dependent pathway. These results suggest that electroacupuncture acts via the vagal nerve and its nicotinic receptor-mediated signaling to inhibit HMGB1 release from ischemic cardiomyocytes. This helps attenuate pro-inflammatory responses and myocardial injury during reperfusion. PMID:26499847

  15. Blood group and serum protein polymorphisms in a population group of Moldavians.

    PubMed

    Varsahr, A M; Scheil, H G; Schmidt, H D

    2006-03-01

    The distribution of the alleles and haplotypes for blood groups A1A2B0, MNSs, RHESUS, P1, KELL-CELLANO and biochemical markers of the alleles of loci AMY2, HPA, GC, C3, TF, BF, CP, PI (including subtypes) were studied in 125 Moldavian individuals from Karahasani settlement, Stefan-Voda District, Republic of Moldavia. The results show that the gene pool of Moldavians is similar to those of Southeastern European populations. PMID:16623088

  16. The quantification of the histochemical protein staining with 2-hydroxy-1-naphthaldehyde (HNA) demonstrating primary amino groups of proteins.

    PubMed

    Nöhammer, G

    1991-01-01

    The usefulness of the HNA-pH4-1d staining, which histochemically demonstrates primary protein amino groups under the conditions used, for the microphotometric quantification of proteins was investigated. A correlation (r = 0.986) has been found between the mean protein contents of fresh frozen and fixed sections prepared from different tissues of rats and the corresponding mean integrated extinction values determined histophotometrically after HNA-pH4-1d staining. A histophotometric extinction of E = 0.284 corresponded to 10(-12) g protein. The mean integrated extinction values determined cytophotometrically of different single cells and nuclei stained using the tetrazonium coupling method for proteins correlated (r = 0.989) with corresponding extinction values measured after HNA-pH4-1d staining. A cytophotometric extinction after HNA-pH4-1d staining of E = 0.130 correspond to 10(-12) g protein. PMID:2048388

  17. Control of nuclear activities by substrate-selective and protein-group SUMOylation.

    PubMed

    Jentsch, Stefan; Psakhye, Ivan

    2013-01-01

    Reversible modification of proteins by SUMO (small ubiquitin-like modifier) affects a large number of cellular processes. In striking contrast to the related ubiquitin pathway, only a few enzymes participate in the SUMO system, although this pathway has numerous substrates as well. Emerging evidence suggests that SUMOylation frequently targets entire groups of physically interacting proteins rather than individual proteins. Protein-group SUMOylation appears to be triggered by recruitment of SUMO ligases to preassembled protein complexes. Because SUMOylation typically affects groups of proteins that bear SUMO-interaction motifs (SIMs), protein-group SUMOylation may foster physical interactions between proteins through multiple SUMO-SIM interactions. Individual SUMO modifications may act redundantly or additively, yet they may mediate dedicated functions as well. In this review, we focus on the unorthodox principles of this pathway and give examples for SUMO-controlled nuclear activities. We propose that collective SUMOylation is typical for nuclear assemblies and argue that SUMO serves as a distinguishing mark for functionally engaged protein fractions. PMID:24016193

  18. IL-1 receptor antagonist (IL-1Ra) does not inhibit the production of C-reactive protein or serum amyloid A protein by human primary hepatocytes. Differential regulation in normal and tumour cells.

    PubMed Central

    Gabay, C; Genin, B; Mentha, G; Iynedjian, P B; Roux-Lombard, P; Guerne, P A

    1995-01-01

    The synthesis of some class 1 acute-phase proteins (APP), including C-reactive protein (CRP) and serum amyloid A (SAA) protein is completely blocked by the IL-1 receptor antagonist (IL-1Ra), whereas the production of fibrinogen, a class 2 APP, is increased by IL-1Ra in hepatoma cells, but this has never been tested in human hepatocytes in primary culture. Since previous studies on the contributions of cytokine inhibitors in connective tissues diseases suggested that IL-1 and tumour necrosis factor-alpha (TNF-alpha) might play an important role in the regulation of CRP, we decided to examine in more detail the respective roles of IL-1 beta, IL-6, and TNF-alpha and their inhibitors in the production of APP by human primary hepatocytes versus the hepatoma cell line PLC/PRF/5. In the hepatoma cell line, IL-1 beta and/or TNF-alpha had synergistic effects with IL-6 on the production of CRP and SAA. In contrast, these cytokines were devoid of effect in normal hepatocytes. The production of fibrinogen was increased by IL-6 and decreased by IL-1 (and TNF-alpha) in both cell types. The secretion of CRP and SAA by primary hepatocytes incubated with a cytokine-rich mononuclear cell-conditioned medium was totally unaffected by IL-1Ra or anti-TNF-alpha antibodies. In contrast, the addition of IL-1Ra increased the production of fibrinogen by both hepatoma cells and primary hepatocytes incubated with the mononuclear cell-conditioned medium. We therefore conclude that IL-1 beta and TNF-alpha do not exert any significant effect on the synthesis of CRP and SAA by human primary hepatocytes. Images Fig. 6 PMID:7743670

  19. Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

    2016-01-01

    We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.

  20. Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies.

    PubMed

    Zhang, Hao; Liu, Haijun; Blankenship, Robert E; Gross, Michael L

    2016-01-01

    We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting. PMID:26384685

  1. Cotranscriptional splicing of a group I intron is facilitated by the Cbp2 protein

    SciTech Connect

    Lewin, A.S.; Thomas, J. Jr.; Tirupati, H.K.

    1995-12-01

    This report investigates the coupling between transcription and splicing of a mitochondrial group I intron in Saccharomyces cerevisiae and the effect of the Cbp2 protein on splicing. 65 refs., 7 figs.

  2. Heterologous expression and functional analysis of the wheat group 1 pathogenesis-related (PR-1) proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The group 1 pathogenesis-related (PR-1) proteins have been widely used as hallmarks of plant defense pathways, but their biological functions are still unknown. We report here the functional analysis of two basic PR-1 proteins following the identification of the wheat PR-1 gene family (Lu et al., 20...

  3. Effects of tyrosine kinase inhibitors and CXCR4 antagonist on tumor growth and angiogenesis in rat glioma model: MRI and protein analysis study.

    PubMed

    Ali, Meser M; Kumar, Sanath; Shankar, Adarsh; Varma, Nadimpalli R S; Iskander, A S M; Janic, Branislava; Chwang, Wilson B; Jain, Rajan; Babajeni-Feremi, Abbas; Borin, Thaiz F; Bagher-Ebadian, Hassan; Brown, Stephen L; Ewing, James R; Arbab, Ali S

    2013-12-01

    The aim of the study was to determine the antiangiogenic efficacy of vatalanib, sunitinib, and AMD3100 in an animal model of human glioblastoma (GBM) by using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and tumor protein expression analysis. Orthotopic GBM-bearing animals were randomly assigned either to control group or vatalanib, sunitinib, and AMD3100 treatment groups. Following 2 weeks of drug treatment, tumor growth and vascular parameters were measured using DCE-MRI. Expression of different angiogenic factors in tumor extracts was measured using a membrane-based human antibody array kit. Tumor angiogenesis and invasion were determined by immunohistochemistry. DCE-MRI showed a significant increase in tumor size after vatalanib treatment. AMD3100-treated group showed a significant decrease in a number of vascular parameters determined by DCE-MRI. AMD3100 significantly decreased the expression of different angiogenic factors compared to sunitinib or vatalanib; however, there were no significant changes in vascular density among the groups. Sunitinib-treated animals showed significantly higher migration of the invasive cells, whereas in both vatalanib- and AMD3100-treated animals the invasive cell migration distance was significantly lower compared to that of control. Vatalanib and sunitinib resulted in suboptimal therapeutic effect, but AMD3100 treatment resulted in a significant reduction in tumor growth, permeability, interstitial space volume, and invasion of tumor cells in an animal model of GBM. PMID:24466368

  4. Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein.

    PubMed

    Buffalo, Cosmo Z; Bahn-Suh, Adrian J; Hirakis, Sophia P; Biswas, Tapan; Amaro, Rommie E; Nizet, Victor; Ghosh, Partho

    2016-01-01

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ∼90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant 'reading head' in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M-C4BP interaction, and also inform a path towards vaccine design. PMID:27595425

  5. Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1).

    PubMed

    Getlik, Matthäus; Smil, David; Zepeda-Velázquez, Carlos; Bolshan, Yuri; Poda, Gennady; Wu, Hong; Dong, Aiping; Kuznetsova, Ekaterina; Marcellus, Richard; Senisterra, Guillermo; Dombrovski, Ludmila; Hajian, Taraneh; Kiyota, Taira; Schapira, Matthieu; Arrowsmith, Cheryl H; Brown, Peter J; Vedadi, Masoud; Al-Awar, Rima

    2016-03-24

    WD repeat-containing protein 5 (WDR5) is an important component of the multiprotein complex essential for activating mixed-lineage leukemia 1 (MLL1). Rearrangement of the MLL1 gene is associated with onset and progression of acute myeloid and lymphoblastic leukemias, and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics. Our previous work showed that binding of small molecule ligands to WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase activity. Initial structure-activity relationship studies identified N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides as potent and selective antagonists of this protein-protein interaction. Guided by crystal structure data and supported by in silico library design, we optimized the scaffold by varying the C-1 benzamide and C-5 substituents. This allowed us to develop the first highly potent (Kdisp < 100 nM) small molecule antagonists of the WDR5-MLL1 interaction and demonstrate that N-(4-(4-methylpiperazin-1-yl)-3'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 16d (OICR-9429) is a potent and selective chemical probe suitable to help dissect the biological role of WDR5. PMID:26958703

  6. Mathematical aspects of molecular replacement. III. Properties of space groups preferred by proteins in the Protein Data Bank.

    PubMed

    Chirikjian, G; Sajjadi, S; Toptygin, D; Yan, Y

    2015-03-01

    The main goal of molecular replacement in macromolecular crystallography is to find the appropriate rigid-body transformations that situate identical copies of model proteins in the crystallographic unit cell. The search for such transformations can be thought of as taking place in the coset space Γ\\G where Γ is the Sohncke group of the macromolecular crystal and G is the continuous group of rigid-body motions in Euclidean space. This paper, the third in a series, is concerned with viewing nonsymmorphic Γ in a new way. These space groups, rather than symmorphic ones, are the most common ones for protein crystals. Moreover, their properties impact the structure of the space Γ\\G. In particular, nonsymmorphic space groups contain both Bieberbach subgroups and symmorphic subgroups. A number of new theorems focusing on these subgroups are proven, and it is shown that these concepts are related to the preferences that proteins have for crystallizing in different space groups, as observed in the Protein Data Bank. PMID:25727867

  7. Effects of surface functional groups on the formation of nanoparticle-protein corona

    PubMed Central

    Podila, R.; Chen, R.; Ke, P. C.; Brown, J. M.; Rao, A. M.

    2012-01-01

    Herein, we examined the dependence of protein adsorption on the nanoparticle surface in the presence of functional groups. Our UV-visible spectrophotometry, transmission electron microscopy, infrared spectroscopy, and dynamic light scattering measurements evidently suggested that the functional groups play an important role in the formation of nanoparticle-protein corona. We found that uncoated and surfactant-free silver nanoparticles derived from a laser ablation process promoted a maximum protein (bovine serum albumin) coating due to increased changes in entropy. On the other hand, bovine serum albumin displayed a relatively lower affinity for electrostatically stabilized nanoparticles due to the constrained entropy changes. PMID:23341687

  8. Kicking against the PRCs – A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2

    PubMed Central

    Perera, Pumi; Mora-García, Santiago; de Leau, Erica; Thornton, Harry; de Alves, Flavia Lima; Rapsilber, Juri; Yang, Suxin; James, Geo Velikkakam; Schneeberger, Korbinian; Finnegan, E. Jean; Turck, Franziska; Goodrich, Justin

    2015-01-01

    The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits Pc

  9. Bayesian identification of protein differential expression in multi-group isobaric labelled mass spectrometry data.

    PubMed

    Jow, Howsun; Boys, Richard J; Wilkinson, Darren J

    2014-10-01

    In this paper we develop a Bayesian statistical inference approach to the unified analysis of isobaric labelled MS/MS proteomic data across multiple experiments. An explicit probabilistic model of the log-intensity of the isobaric labels' reporter ions across multiple pre-defined groups and experiments is developed. This is then used to develop a full Bayesian statistical methodology for the identification of differentially expressed proteins, with respect to a control group, across multiple groups and experiments. This methodology is implemented and then evaluated on simulated data and on two model experimental datasets (for which the differentially expressed proteins are known) that use a TMT labelling protocol. PMID:25153608

  10. Quantification of protein group coherence and pathway assignment using functional association

    PubMed Central

    2011-01-01

    Background Genomics and proteomics experiments produce a large amount of data that are awaiting functional elucidation. An important step in analyzing such data is to identify functional units, which consist of proteins that play coherent roles to carry out the function. Importantly, functional coherence is not identical with functional similarity. For example, proteins in the same pathway may not share the same Gene Ontology (GO) terms, but they work in a coordinated fashion so that the aimed function can be performed. Thus, simply applying existing functional similarity measures might not be the best solution to identify functional units in omics data. Results We have designed two scores for quantifying the functional coherence by considering association of GO terms observed in two biological contexts, co-occurrences in protein annotations and co-mentions in literature in the PubMed database. The counted co-occurrences of GO terms were normalized in a similar fashion as the statistical amino acid contact potential is computed in the protein structure prediction field. We demonstrate that the developed scores can identify functionally coherent protein sets, i.e. proteins in the same pathways, co-localized proteins, and protein complexes, with statistically significant score values showing a better accuracy than existing functional similarity scores. The scores are also capable of detecting protein pairs that interact with each other. It is further shown that the functional coherence scores can accurately assign proteins to their respective pathways. Conclusion We have developed two scores which quantify the functional coherence of sets of proteins. The scores reflect the actual associations of GO terms observed either in protein annotations or in literature. It has been shown that they have the ability to accurately distinguish biologically relevant groups of proteins from random ones as well as a good discriminative power for detecting interacting pairs of

  11. Ethrel-stimulated prolongation of latex flow in the rubber tree (Hevea brasiliensis Muell. Arg.): an Hev b 7-like protein acts as a universal antagonist of rubber particle aggregating factors from lutoids and C-serum.

    PubMed

    Shi, Min-Jing; Cai, Fu-Ge; Tian, Wei-Min

    2016-02-01

    Ethrel is the most effective stimuli in prolonging the latex flow that consequently increases yield per tapping. This effect is largely ascribed to the enhanced lutoid stability, which is associated with the decreased release of initiators of rubber particle (RP) aggregation from lutoid bursting. However, the increase in both the bursting index of lutoids and the duration of latex flow after applying ethrel or ethylene gas in high concentrations suggests that a new mechanism needs to be introduced. In this study, a latex allergen Hev b 7-like protein in C-serum was identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS). In vitro analysis showed that the protein acted as a universal antagonist of RP aggregating factors from lutoids and C-serum. Ethrel treatment obviously weakened the effect of C-serum on RP aggregation, which was closely associated with the increase in the level of the Hev b 7-like protein and the decrease in the level of the 37 kDa protein, as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting analysis and antibody neutralization. Thus, the increase of the Hev b 7-like protein level or the ratio of the Hev b 7-like protein to the 37 kDa protein in C-serum should be primarily ascribed to the ethrel-stimulated prolongation of latex flow duration. PMID:26381537

  12. Contribution of polar groups in the interior of a protein to the conformational stability.

    PubMed

    Takano, K; Yamagata, Y; Yutani, K

    2001-04-17

    It has been generally believed that polar residues are usually located on the surface of protein structures. However, there are many polar groups in the interior of the structures in reality. To evaluate the contribution of such buried polar groups to the conformational stability of a protein, nonpolar to polar mutations (L8T, A9S, A32S, I56T, I59T, I59S, A92S, V93T, A96S, V99T, and V100T) in the interior of a human lysozyme were examined. The thermodynamic parameters for denaturation were determined using a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. If a polar group had a heavy energy cost to be buried, a mutant protein would be remarkably destabilized. However, the stability (Delta G) of the Ala to Ser and Val to Thr mutant human lysozymes was comparable to that of the wild-type protein, suggesting a low-energy penalty of buried polar groups. The structural analysis showed that all polar side chains introduced in the mutant proteins were able to find their hydrogen bond partners, which are ubiquitous in protein structures. The empirical structure-based calculation of stability change (Delta Delta G) [Takano et al. (1999) Biochemistry 38, 12698--12708] revealed that the mutant proteins decreased the hydrophobic effect contributing to the stability (Delta G(HP)), but this destabilization was recovered by the hydrogen bonds newly introduced. The present study shows the favorable contribution of polar groups with hydrogen bonds in the interior of protein molecules to the conformational stability. PMID:11294653

  13. Antagonistic formation motion of cooperative agents

    NASA Astrophysics Data System (ADS)

    Lu, Wan-Ting; Dai, Ming-Xiang; Xue, Fang-Zheng

    2015-02-01

    This paper investigates a new formation motion problem of a class of first-order multi-agent systems with antagonistic interactions. A distributed formation control algorithm is proposed for each agent to realize the antagonistic formation motion. A sufficient condition is derived to ensure that all of the agents make an antagonistic formation motion in a distributed manner. It is shown that all of the agents can be spontaneously divided into several groups and that agents in the same group collaborate while agents in different groups compete. Finally, a numerical simulation is included to demonstrate our theoretical results. Project supported by the National Natural Science Foundation of China (Grant Nos. 61203080 and 61473051) and the Natural Science Foundation of Chongqing City (Grant No. CSTC 2011BB0081).

  14. Mapping functional group free energy patterns at protein occluded sites: nuclear receptors and G-protein coupled receptors.

    PubMed

    Lakkaraju, Sirish Kaushik; Yu, Wenbo; Raman, E Prabhu; Hershfeld, Alena V; Fang, Lei; Deshpande, Deepak A; MacKerell, Alexander D

    2015-03-23

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents. PMID:25692383

  15. Mapping Functional Group Free Energy Patterns at Protein Occluded Sites: Nuclear Receptors and G-Protein Coupled Receptors

    PubMed Central

    2015-01-01

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents. PMID:25692383

  16. Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group.

    PubMed

    Lim, Woon Ki; Rösgen, Jörg; Englander, S Walter

    2009-02-24

    The mechanism by which urea and guanidinium destabilize protein structure is controversial. We tested the possibility that these denaturants form hydrogen bonds with peptide groups by measuring their ability to block acid- and base-catalyzed peptide hydrogen exchange. The peptide hydrogen bonding found appears sufficient to explain the thermodynamic denaturing effect of urea. Results for guanidinium, however, are contrary to the expectation that it might H-bond. Evidently, urea and guanidinium, although structurally similar, denature proteins by different mechanisms. PMID:19196963

  17. Quantitative toxicoproteomic analysis of zebrafish embryos exposed to a retinoid X receptor antagonist UVI3003.

    PubMed

    Zheng, Liang; Yu, Jianlan; Shi, Huahong; Xia, Liang; Xin, Qi; Zhang, Qiang; Zhao, Heng; Luo, Ji; Jin, Wenhai; Li, Daoji; Zhou, Junliang

    2015-09-01

    Retinoid X receptor (RXR) antagonists, including some environmental endocrine disruptors, have a teratogenic effect on vertebrate embryos. To investigate the toxicological mechanism on the protein expression level, a quantitative proteomic study was conducted to analyze the proteome alterations of zebrafish (Danio rerio) embryos exposed to gradient concentrations of a representative RXR antagonist UVI3003. Using isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labeling coupled nano high-performance liquid chromatography-tandem mass spectrometry (nano HPLC-MS/MS), in total 6592 proteins were identified, among which 195 proteins were found to be differentially expressed by more than a two-fold change in exposed groups compared with the control. Gene ontology analysis showed that these differential proteins were mostly involved in anatomical structure development, biosynthetic process, ion binding and oxidoreductase activity. Moreover, the biological pathways of translation, lipoprotein metabolism, cell survival and gluconeogenesis were intensively inhibited after exposure. Some significantly downregulated proteins such as apolipoprotein A-I and vitellogenin and upregulated proteins such as calcium activated nucleotidase 1b, glutathione S-transferase and glucose 6-dehydrogenases showed a strong dose-dependent response. The results provided new insight into the molecular details of RXR antagonist-induced teratogenicity and added novel information of pathways and potential biomarkers for evaluation of RXR interfering activity. PMID:25581642

  18. Cloning, sequencing, and expression of a fibronectin/fibrinogen-binding protein from group A streptococci.

    PubMed Central

    Courtney, H S; Li, Y; Dale, J B; Hasty, D L

    1994-01-01

    Lipoteichoic acid and several streptococcal proteins have been reported to bind fibronectin (Fn) or fibrinogen (Fgn), which may serve as host receptors. We searched for such proteins by screening a library of genes from M type 5 group A streptococci cloned into Escherichia coli. Lysates of clones were probed with biotinylated Fn and biotinylated Fgn. One clone expressed a 54-kDa protein that reacted with Fn and Fgn. The protein, termed FBP54, was purified and used to immunize rabbits. Anti-FBP54 serum reacted with purified, recombinant FBP54 and with a protein of similar electrophoretic mobility in extracts of M type 5, 6, and 24 streptococci. Anti-FBP54 serum also reacted with 5 of 15 strains of intact, live streptococci, suggesting that FBP54 may be a surface antigen. Southern blot analysis confirmed that the gene is found in group A streptococci but not in Staphylococcus aureus or E. coli. The cloned gene was sequenced and contained an open reading frame encoding a protein with a calculated molecular weight of 54,186. Partial amino acid sequencing of purified FBP54 confirmed that this open reading frame encoded the protein. As determined by utilizing fusion proteins containing truncated forms of FBP54, the primary Fn/Fgn-binding domain appears to be contained in residues 1 to 89. These data suggest that FBP54 may be a surface protein of streptococci that reacts with both Fn and Fgn and therefore may participate in the adhesion of group A streptococci to host cells. Images PMID:8063411

  19. Polycomb Group (PcG) Proteins and Human Cancers: Multifaceted Functions and Therapeutic Implications

    PubMed Central

    Wang, Wei; Qin, Jiang-Jiang; Voruganti, Sukesh; Nag, Subhasree; Zhou, Jianwei; Zhang, Ruiwen

    2016-01-01

    Polycomb group (PcG) proteins are transcriptional repressors that regulate several crucial developmental and physiological processes in the cell. More recently, they have been found to play important roles in human carcinogenesis and cancer development and progression. The deregulation and dysfunction of PcG proteins often lead to blocking or inappropriate activation of developmental pathways, enhancing cellular proliferation, inhibiting apoptosis, and increasing the cancer stem cell population. Genetic and molecular investigations of PcG proteins have long been focused on their PcG functions. However, PcG proteins have recently been shown to exert non-polycomb functions, contributing to the regulation of diverse cellular functions. We and others have demonstrated that PcG proteins regulate the expression and function of several oncogenes and tumor suppressor genes in a PcG-independent manner, and PcG proteins are associated with the survival of patients with cancer. In this review, we summarize the recent advances in the research on PcG proteins, including both the polycomb-repressive and non-polycomb functions. We specifically focus on the mechanisms by which PcG proteins play roles in cancer initiation, development, and progression. Finally, we discuss the potential value of PcG proteins as molecular biomarkers for the diagnosis and prognosis of cancer, and as molecular targets for cancer therapy. PMID:26227500

  20. Suppression of Host Gene Expression by nsp1 Proteins of Group 2 Bat Coronaviruses ▿

    PubMed Central

    Tohya, Yukinobu; Narayanan, Krishna; Kamitani, Wataru; Huang, Cheng; Lokugamage, Kumari; Makino, Shinji

    2009-01-01

    nsp1 protein of severe acute respiratory syndrome coronavirus (SARS-CoV), a group 2b CoV, suppresses host gene expression by promoting host mRNA degradation and translation inhibition. The present study analyzed the activities of nsp1 proteins from the group 2 bat CoV strains Rm1, 133, and HKU9-1, belonging to groups 2b, 2c, and 2d, respectively. The host mRNA degradation and translational suppression activities of nsp1 of SARS-CoV and Rm1 nsp1 were similar and stronger than the activities of the nsp1 proteins of 133 and HKU9-1. Rm1 nsp1 expression in trans strongly inhibited the induction of type I interferon (IFN-I) and IFN-stimulated genes in cells infected with an IFN-inducing SARS-CoV mutant, while 133 and HKU9-1 nsp1 proteins had relatively moderate IFN-inhibitory activities. The results of our studies suggested a conserved function among nsp1 proteins of SARS-CoV and group 2 bat CoVs. PMID:19264783

  1. Calcium-dependent ADP-ribosylation of high-mobility-group I (HMGI) proteins.

    PubMed Central

    Giancotti, V; Bandiera, A; Sindici, C; Perissin, L; Crane-Robinson, C

    1996-01-01

    Micrococcal nuclease digestion of nuclei from mouse Lewis lung carcinoma cells releases a protein mixture into the supernatant that lacks histone H1 and contains a full complement of high-mobility-group I (HMGI) proteins (i.e. I, Y and I-C). This implies that all three HMGI proteins are localized at the nuclease-sensitive regions of active chromatin. It is also shown that if Ca2+ ions are present in the nuclear incubation buffer (with or without exogenous nuclease), all three HMGI proteins become ADP-ribosylated. We propose that this modification of HMGI family proteins is part of the general poly(ADP-ribosyl)ation that accompanies DNA damage in apoptosis and other processes. PMID:8760375

  2. Functional gene group analysis indicates no role for heterotrimeric G proteins in cognitive ability.

    PubMed

    Hill, W David; de Leeuw, Christiaan; Davies, Gail; Liewald, David Cherry McLachlan; Payton, Anthony; Craig, Leone C A; Whalley, Lawrence J; Horan, Mike; Ollier, William; Starr, John M; Pendleton, Neil; Posthuma, Danielle; Bates, Timothy C; Deary, Ian J

    2014-01-01

    Previous functional gene group analyses implicated common single nucleotide polymorphisms (SNPs) in heterotrimeric G protein coding genes as being associated with differences in human intelligence. Here, we sought to replicate this finding using five independent cohorts of older adults including current IQ and childhood IQ, and using both gene- and SNP-based analytic strategies. No significant associations were found between variation in heterotrimeric G protein genes and intelligence in any cohort at either of the two time points. These results indicate that, whereas G protein systems are important in cognition, common genetic variation in these genes is unlikely to be a substantial influence on human intelligence differences. PMID:24626473

  3. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini

    PubMed Central

    Tatham, Michael H.; Plechanovová, Anna; Jaffray, Ellis G.; Salmen, Helena; Hay, Ronald T.

    2013-01-01

    The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13–UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation. PMID:23560854

  4. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini.

    PubMed

    Tatham, Michael H; Plechanovová, Anna; Jaffray, Ellis G; Salmen, Helena; Hay, Ronald T

    2013-07-01

    The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13-UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation. PMID:23560854

  5. Serum protein and erythrocyte enzyme polymorphisms in twelve population groups of Hungary.

    PubMed

    Goedde, H W; Czeizel, A; Benkmann, H G; Hummel, K; Fukshansky, N; Kriese, L; Wimmer, U; Gaulke, R; Béres, J; Matsumoto, H

    1995-06-01

    The distribution of the serum proteins C3, TF, HP, GC, BF, AMY2, PLG, GM, and KM and the erythrocyte enzyme polymorphisms GLO, GPT, ESD, ACP, 6-PGD, ADA, AK, PGM1 and PGP amongst twelve population groups in Hungary was investigated. Gene frequencies and genetic distances are discussed in relation to the present geographical locations of these groups and their probable history of migration. PMID:7668845

  6. High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors.

    PubMed Central

    Zwilling, S; König, H; Wirth, T

    1995-01-01

    The octamer transcription factors Oct1 and Oct2 are involved in the transcriptional regulation of both lymphoid-specific and ubiquitously expressed genes. Their activity depends critically on their interaction with distinct cellular cofactors. Therefore, we have isolated cDNAs encoding proteins that physically interact with Oct2. Here we describe the analysis of one such clone, representing the murine homologue of high mobility group (HMG) protein 2. We have mapped the interaction domains for both proteins and have shown that HMG2 and Oct2 interact via their HMG domains and POU homeodomains, respectively. This interaction is not restricted to Oct2, as other members of the octamer transcription factor family like Oct1 and Oct6 also interact with HMG2. The interaction with HMG2 results in a marked increase in the sequence-specific DNA binding activity of the Oct proteins. Interestingly, the HMG2 protein is not present in the protein-DNA complex detected by an electrophoretic mobility shift assay. The Oct and HMG2 proteins also interact in vivo. A chimeric protein, in which the strong transactivation domain of VP16 was fused directly to the HMG domains of HMG2, stimulated the activity of an octamer-dependent reporter construct upon cotransfection. Furthermore, the expression of antisense RNA for HMG2 specifically reduces octamer-dependent transcription. These results suggest that one of the functions of HMG2 is to support the octamer transcription factors in their role as transcriptional activators. Images PMID:7720710

  7. Calcium channel antagonists and the treatment of migraine.

    PubMed

    Greenberg, D A

    1986-01-01

    Despite ongoing dispute over the pathophysiologic basis of migraine, the vasospastic theory of pathogenesis has brought to the forefront a promising class of new antimigraine agents, the Ca2+ channel antagonists. Voltage-dependent Ca2+ channels, integral membrane proteins that permit extracellular Ca2+ to enter cells down their electrical and concentration gradients, have a universal role in stimulus-response coupling in excitable cells. Thus, they participate in translating electrical excitation into secretory and contractile events. Ca2+ channel antagonists, a structurally diverse group of organic compounds, inhibit ion flux through voltage-dependent Ca2+ channels by binding to specific, channel-associated drug receptor sites and thereby reduce the frequency of channel opening in response to membrane depolarization. Ca2+ channels in cardiac muscle, smooth muscle, and neurons all exhibit high affinity for Ca2+ channel antagonists, although neurons also contain a population of drug-resistant channels. Extensive clinical experience in the use of Ca2+ channel antagonists has accumulated from their application to nonneurologic, especially cardiovascular, disorders. Three such drugs, nifedipine, verapamil, and diltiazem, are currently available in the United States, although none are specifically approved for use in migraine. Other agents, such as nimodipine, are likely to be released in the near future. A large number of clinical studies have now addressed the efficacy of Ca2+ channel antagonists in the prophylaxis of migraine headache. Dihydropyridines (nifedipine and nimodipine), phenylalkylamines (verapamil), diphenylalkylamines (flunarizine), and benzothiazepines (diltiazem) have all been examined, and a beneficial effect has been noted in each case. The limited directly comparative data currently available and the difficulties involved in comparing the results of different studies do not presently support claims of superiority for any single agent. This is an

  8. Reptin and Pontin function antagonistically with PcG and TrxG complexes to mediate Hox gene control

    PubMed Central

    Diop, Soda Balla; Bertaux, Karine; Vasanthi, Dasari; Sarkeshik, Ali; Goirand, Benjamin; Aragnol, Denise; Tolwinski, Nicholas S; Cole, Michael D; Pradel, Jacques; Yates, John R; Mishra, Rakesh K; Graba, Yacine; Saurin, Andrew J

    2008-01-01

    Pontin (Pont) and Reptin (Rept) are paralogous ATPases that are evolutionarily conserved from yeast to human. They are recruited in multiprotein complexes that function in various aspects of DNA metabolism. They are essential for viability and have antagonistic roles in tissue growth, cell signalling and regulation of the tumour metastasis suppressor gene, KAI1, indicating that the balance of Pont and Rept regulates epigenetic programmes critical for development and cancer progression. Here, we describe Pont and Rept as antagonistic mediators of Drosophila Hox gene transcription, functioning with Polycomb group (PcG) and Trithorax group proteins to maintain correct patterns of expression. We show that Rept is a component of the PRC1 PcG complex, whereas Pont purifies with the Brahma complex. Furthermore, the enzymatic functions of Rept and Pont are indispensable for maintaining Hox gene expression states, highlighting the importance of these two antagonistic factors in transcriptional output. PMID:18259215

  9. Vasopressin receptor antagonists.

    PubMed

    Palmer, Biff F

    2015-01-01

    Arginine vasopressin (AVP) is the principal hormone involved in regulating the tonicity of body fluids. Less appreciated is the role that AVP plays in a variety of other physiologic functions including glucose metabolism, cardiovascular homeostasis, bone metabolism, and cognitive behavior. AVP receptor antagonists are now available and currently approved to treat hyponatremia. There is a great deal of interest in exploring the potential benefits that these drugs may play in blocking AVP-mediated effects in other organ systems. The purpose of this report is to provide an update on the expanding role of AVP receptor antagonists and what disease states these drugs may eventually be used for. PMID:25604388

  10. A novel integrin {alpha}5{beta}1 antagonistic peptide, A5-1, screened by Protein Chip system as a potent angiogenesis inhibitor

    SciTech Connect

    Kim, Eung-Yoon; Bang, Ji Young; Chang, Soo-Ik; Kang, In-Cheol

    2008-12-26

    Integrin {alpha}5{beta}1 immobilized on a ProteoChip was used to screen new antagonistic peptides from multiple hexapeptide sub-libraries of the positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin {alpha}5{beta}1-Fibronectin interaction was demonstrated on the chip. A novel peptide ligand, A5-1 (VILVLF), with high affinity to integrin {alpha}5{beta}1 was identified from the hexapeptide libraries with this chip-based screening method on the basis of a competitive inhibition assay. A5-1 inhibits the integrin-fibronectin interaction in a dose-dependent manner (IC{sub 50}; 1.56 {+-} 0.28 {mu}M. In addition, it inhibits human umbilical vein endothelial cell proliferation, migration, adhesion, tubular network formation, and bFGF-induced neovascularization in a chick chorioallantoic membrane. These results suggest that A5-1 will be a potent inhibitor of neovascularization.

  11. Endothelin receptors and their antagonists.

    PubMed

    Maguire, Janet J; Davenport, Anthony P

    2015-03-01

    All three members of the endothelin (ET) family of peptides, ET-1, ET-2, and ET-3, are expressed in the human kidney, with ET-1 being the predominant isoform. ET-1 and ET-2 bind to two G-protein-coupled receptors, ETA and ETB, whereas at physiological concentrations ET-3 has little affinity for the ET(A) receptor. The human kidney is unusual among the peripheral organs in expressing a high density of ET(B). The renal vascular endothelium only expresses the ET(B) subtype and ET-1 acts in an autocrine or paracrine manner to release vasodilators. Endothelial ETB in kidney, as well as liver and lungs, also has a critical role in scavenging ET-1 from the plasma. The third major function is ET-1 activation of ET(B) in in the nephron to reduce salt and water re-absorption. In contrast, ET(A) predominate on smooth muscle, causing vasoconstriction and mediating many of the pathophysiological actions of ET-1. The role of the two receptors has been delineated using highly selective ET(A) (BQ123, TAK-044) and ET(B) (BQ788) peptide antagonists. Nonpeptide antagonists, bosentan, macitentan, and ambrisentan, that are either mixed ET(A)/ET(B) antagonists or display ET(A) selectivity, have been approved for clinical use but to date are limited to pulmonary hypertension. Ambrisentan is in clinical trials in patients with type 2 diabetic nephropathy. This review summarizes ET-receptor antagonism in the human kidney, and considers the relative merits of selective versus nonselective antagonism in renal disease. PMID:25966344

  12. Updated model of group A Streptococcus M proteins based on a comprehensive worldwide study.

    PubMed

    McMillan, D J; Drèze, P-A; Vu, T; Bessen, D E; Guglielmini, J; Steer, A C; Carapetis, J R; Van Melderen, L; Sriprakash, K S; Smeesters, P R

    2013-05-01

    Group A Streptococcus (GAS) M protein is an important virulence factor and potential vaccine antigen, and constitutes the basis for strain typing (emm-typing). Although >200 emm-types are characterized, structural data were obtained from only a limited number of emm-types. We aim to evaluate the sequence diversity of near-full-length M proteins from worldwide sources and analyse their structure, sequence conservation and classification. GAS isolates recovered from throughout the world during the last two decades underwent emm-typing and complete emm gene sequencing. Predicted amino acid sequence analyses, secondary structure predictions and vaccine epitope mapping were performed using MUSCLE and Geneious software. A total of 1086 isolates from 31 countries were analysed, representing 175 emm-types. emm-type is predictive of the whole protein structure, independent of geographical origin or clinical association. Findings of an emm-type paired with multiple, highly divergent central regions were not observed. M protein sequence length, the presence or absence of sequence repeats and predicted secondary structure were assessed in the context of the latest vaccine developments. Based on these global data, the M6 protein model is updated to a three representative M protein (M5, M80 and M77) model, to aid in epidemiological analysis, vaccine development and M protein-related pathogenesis studies. PMID:23464795

  13. Opioid Antagonist Impedes Exposure.

    ERIC Educational Resources Information Center

    Merluzzi, Thomas V.; And Others

    1991-01-01

    Thirty spider-phobic adults underwent exposure to 17 phobic-related, graded performance tests. Fifteen subjects were assigned to naltrexone, an opioid antagonist, and 15 were assigned to placebo. Naltrexone had a significant effect on exposure, with naltrexone subjects taking significantly longer to complete first 10 steps of exposure and with…

  14. Spiropiperidine CCR5 antagonists.

    PubMed

    Rotstein, David M; Gabriel, Stephen D; Makra, Ferenc; Filonova, Lubov; Gleason, Shelley; Brotherton-Pleiss, Christine; Setti, Lina Q; Trejo-Martin, Alejandra; Lee, Eun Kyung; Sankuratri, Surya; Ji, Changhua; Derosier, Andre; Dioszegi, Marianna; Heilek, Gabrielle; Jekle, Andreas; Berry, Pamela; Weller, Paul; Mau, Cheng-I

    2009-09-15

    A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed. PMID:19674898

  15. Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?

    PubMed

    Sedlák, Erik; Stagg, Loren; Wittung-Stafshede, Pernilla

    2008-11-01

    We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge -19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT trs/d[ion] (T trs; thermal midpoint). We show that dT trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins. PMID:18782555

  16. Hydrogen bonding motifs of protein side chains: descriptions of binding of arginine and amide groups.

    PubMed Central

    Shimoni, L.; Glusker, J. P.

    1995-01-01

    The modes of hydrogen bonding of arginine, asparagine, and glutamine side chains and of urea have been examined in small-molecule crystal structures in the Cambridge Structural Database and in crystal structures of protein-nucleic acid and protein-protein complexes. Analysis of the hydrogen bonding patterns of each by graph-set theory shows three patterns of rings (R) with one or two hydrogen bond acceptors and two donors and with eight, nine, or six atoms in the ring, designated R2(2)(8), R2(2)(9), and R1(2)(6). These three patterns are found for arginine-like groups and for urea, whereas only the first two patterns R2(2)(8) and R2(2)(9) are found for asparagine- and glutamine-like groups. In each case, the entire system is planar within 0.7 A or less. On the other hand, in macromolecular crystal structures, the hydrogen bonding patterns in protein-nucleic acid complexes between the nucleic acid base and the protein are all R2(2)(9), whereas hydrogen bonding between Watson-Crick-like pairs of nucleic acid bases is R2(2)(8). These two hydrogen bonding arrangements [R2(2)(9)] and R2(2)(8)] are predetermined by the nature of the groups available for hydrogen bonding. The third motif identified, R1(2)(6), involves hydrogen bonds that are less linear than in the other two motifs and is found in proteins. PMID:7773178

  17. Prediction of functional sites in proteins using conserved functional group analysis.

    PubMed

    Innis, C Axel; Anand, A Prem; Sowdhamini, R

    2004-04-01

    A detailed knowledge of a protein's functional site is an absolute prerequisite for understanding its mode of action at the molecular level. However, the rapid pace at which sequence and structural information is being accumulated for proteins greatly exceeds our ability to determine their biochemical roles experimentally. As a result, computational methods are required which allow for the efficient processing of the evolutionary information contained in this wealth of data, in particular that related to the nature and location of functionally important sites and residues. The method presented here, referred to as conserved functional group (CFG) analysis, relies on a simplified representation of the chemical groups found in amino acid side-chains to identify functional sites from a single protein structure and a number of its sequence homologues. We show that CFG analysis can fully or partially predict the location of functional sites in approximately 96% of the 470 cases tested and that, unlike other methods available, it is able to tolerate wide variations in sequence identity. In addition, we discuss its potential in a structural genomics context, where automation, scalability and efficiency are critical, and an increasing number of protein structures are determined with no prior knowledge of function. This is exemplified by our analysis of the hypothetical protein Ydde_Ecoli, whose structure was recently solved by members of the North East Structural Genomics consortium. Although the proposed active site for this protein needs to be validated experimentally, this example illustrates the scope of CFG analysis as a general tool for the identification of residues likely to play an important role in a protein's biochemical function. Thus, our method offers a convenient solution to rapidly and automatically process the vast amounts of data that are beginning to emerge from structural genomics projects. PMID:15033369

  18. High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

    PubMed Central

    Singh, Indrabahadur; Ozturk, Nihan; Cordero, Julio; Mehta, Aditi; Hasan, Diya; Cosentino, Claudia; Sebastian, Carlos; Krüger, Marcus; Looso, Mario; Carraro, Gianni; Bellusci, Saverio; Seeger, Werner; Braun, Thomas; Mostoslavsky, Raul; Barreto, Guillermo

    2015-01-01

    The eukaryotic genome is organized into chromatins, the physiological template for DNA-dependent processes including replication, recombination, repair, and transcription. Chromatin-mediated transcription regulation involves DNA methylation, chromatin remodeling, and histone modifications. However, chromatin also contains non-histone chromatin-associated proteins, of which the high-mobility group (HMG) proteins are the most abundant. Although it is known that HMG proteins induce structural changes of chromatin, the processes underlying transcription regulation by HMG proteins are poorly understood. Here we decipher the molecular mechanism of transcription regulation mediated by the HMG AT-hook 2 protein (HMGA2). We combined proteomic, ChIP-seq, and transcriptome data to show that HMGA2-induced transcription requires phosphorylation of the histone variant H2AX at S139 (H2AXS139ph; γ-H2AX) mediated by the protein kinase ataxia telangiectasia mutated (ATM). Furthermore, we demonstrate the biological relevance of this mechanism within the context of TGFβ1 signaling. The interplay between HMGA2, ATM, and H2AX is a novel mechanism of transcription initiation. Our results link H2AXS139ph to transcription, assigning a new function for this DNA damage marker. Controlled chromatin opening during transcription may involve intermediates with DNA breaks that may require mechanisms that ensure the integrity of the genome. PMID:26045162

  19. Genetic linkage of capsid protein-encoding RNA segments in group A equine rotaviruses.

    PubMed

    Miño, Samuel; Barrandeguy, María; Parreño, Viviana; Parra, Gabriel I

    2016-04-01

    Rotavirus virions are formed by three concentric protein layers that enclose the 11 dsRNA genome segments and the viral proteins VP1 and VP3. Interactions amongst the capsid proteins (VP2, VP6, VP7 and VP4) have been described to play a major role in viral fitness, whilst restricting the reassortment of the genomic segments during co-infection with different rotavirus strains. In this work we describe and characterize the linkage between VP6 and VP7 proteins based on structural and genomic analyses of group A rotavirus strains circulating in Argentinean horses. Strains with the VP7 genotype G3 showed a strong association with the VP6 genotype I6, whilst strains with G14 were associated with the I2 genotype. Most of the differences on the VP6 and VP7 proteins were observed in interactive regions between the two proteins, suggesting that VP6 : VP7 interactions may drive the co-evolution and co-segregation of their respective gene segments. PMID:26758293

  20. Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

    PubMed

    Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

    1990-03-01

    We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis. PMID:2106493

  1. Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

    PubMed Central

    Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

    1990-01-01

    We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis. Images PMID:2106493

  2. Xanthines as Adenosine Receptor Antagonists

    PubMed Central

    Jacobson, Kenneth A.

    2013-01-01

    The natural plant alkaloids caffeine and theophylline were the first adenosine receptor (AR) antagonists described in the literature. They exhibit micromolar affinities and are non-selective. A large number of derivatives and analogs have subsequently been synthesized and evaluated as AR antagonists. Very potent antagonists have thus been developed with selectivity for each of the four AR subtypes. PMID:20859796

  3. Behavioral effects of nicotinic antagonist mecamylamine in a rat model of depression: prefrontal cortex level of BDNF protein and monoaminergic neurotransmitters.

    PubMed

    Aboul-Fotouh, Sawsan

    2015-03-01

    Several studies have pointed to the nicotinic acetylcholine receptor (nAChR) antagonists, such as mecamylamine (MEC), as a potential therapeutic target for the treatment of depression. The present study evaluated the behavioral and neurochemical effects of chronic administration of MEC (1, 2, and 4 mg/kg/day, intraperitoneally (i.p.)) in Wistar rats exposed to chronic restraint stress (CRS, 4 h × 6 W). MEC prevented CRS-induced depressive-like behavior via increasing sucrose preference, body weight, and forced swim test (FST) struggling and swimming while reducing immobility in FST and hypothalamic-pituitary-adrenal (HPA) axis hyperactivity (adrenal gland weight and serum corticosterone). At the same time, MEC amended CRS-induced anxiety as indicated by decreasing central zone duration in open field test and increasing active interaction duration. Additionally, MEC modulated the prefrontal cortex (PFC) level of brain-derived neurotrophic factor (BDNF), 5-hydroxy tryptamine (5-HT), and norepinephrine (NE). In conclusion, the present data suggest that MEC possesses antidepressant and anxiolytic-like activities in rats exposed to CRS. These behavioral effects may be in part mediated by reducing HPA axis hyperactivity and increasing PFC level of BDNF and monoamines. Accordingly, these findings further support the hypothesis that nAChRs blockade might afford a novel promising strategy for pharmacotherapy of depression. PMID:25315361

  4. The Discovery of a Potent Small-Molecule Antagonist of Inhibitor of Apoptosis (IAP) Proteins and Clinical Candidate for the Treatment of Cancer (GDC-0152)

    PubMed Central

    Flygare, John A.; Beresini, Maureen; Budha, Nageshwar; Chan, Helen; Chan, Iris T.; Cheeti, Sravanthi; Cohen, Frederick; Deshayes, Kurt; Doerner, Karl; Eckhardt, S. Gail; Elliott, Linda O.; Feng, Bainian; Franklin, Matthew C.; Reisner, Stacy Frankovitz; Gazzard, Lewis; Halladay, Jason; Hymowitz, Sarah G.; La, Hank; LoRusso, Patricia; Maurer, Brigitte; Murray, Lesley; Plise, Emile; Quan, Clifford; Stephan, Jean-Philippe; Shin, Young G.; Tom, Jeffrey; Tsui, Vickie; Um, Joanne; Varfolomeev, Eugene; Vucic, Domagoj; Wagner, Andrew J.; Wallweber, Heidi J.A.; Wang, Lan; Ware, Joseph; Wen, Zhaoyang; Wong, Harvey; Wong, Jonathan M.; Wong, Melisa; Wong, Susan; Yu, Ron; Zobel, Kerry; Fairbrother, Wayne J.

    2012-01-01

    A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with Ki values of 28, 14, 17 and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Compound 1 inhibits tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human clinical trials and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance in humans was 9 ± 3 mL/min/kg and volume of distribution was 0.6 ± 0.2 L/kg. PMID:22413863

  5. Conformational relaxation and water penetration coupled to ionization of internal groups in proteins.

    PubMed

    Damjanović, Ana; Brooks, Bernard R; García-Moreno, Bertrand

    2011-04-28

    Molecular dynamics simulations were used to examine the effects of ionization of internal groups on the structures of eighteen variants of staphylococcal nuclease (SNase) with internal Lys, Asp, or Glu. In most cases the RMSD values of internal ionizable side chains were larger when the ionizable moieties were charged than when they were neutral. Calculations of solvent-accessible surface area showed that the internal ionizable side chains were buried in the protein interior when they were neutral and moved toward crevices and toward the protein-water interface when they were charged. The only exceptions are Lys-36, Lys-62, and Lys-103, which remained buried even after charging. With the exception of Lys-38, the number of internal water molecules surrounding the ionizable group increased upon charging: the average number of water oxygen atoms within the first hydration shell increased by 1.7 for Lys residues, by 5.2 for Asp residues, and by 3.2 for Glu residues. The polarity of the microenvironment of the ionizable group also increased when the groups were charged: the average number of polar atoms of any kind within the first hydration shell increased by 2.7 for Lys residues, by 4.8 for Asp residues, and by 4.0 for Glu residues. An unexpected correlation was observed between the absolute value of the shifts in pK(a) values measured experimentally, and several parameters of structural relaxation: the net difference in the polarity of the microenvironment of the charged and neutral forms of the ionizable groups, the net difference in hydration of the charged and neutral forms of the ionizable groups, and the difference in RMSD values of the charged and neutral forms of the ionizable groups. The effects of ionization of internal groups on the conformation of the backbone were noticeable but mostly small and localized to the area immediately next to the internal ionizable moiety. Some variants did exhibit local unfolding. PMID:21428436

  6. Polycomb Group Proteins: Multi-Faceted Regulators of Somatic Stem Cells and Cancer

    PubMed Central

    Sauvageau, Martin; Sauvageau, Guy

    2016-01-01

    Polycomb Group (PcG) proteins are transcriptional repressors that epigenetically modify chromatin and participate in the establishment and maintenance of cell fates. These proteins play important roles in both stem cell self-renewal and in cancer development. Our understanding of their mechanism of action has greatly advanced over the past 10 years, but many unanswered questions remain. In this review, we present the currently available experimental data that connect PcG protein function with some of the key processes which govern somatic stem cell activity. We also highlight recent studies suggesting that a delicate balance in PcG gene dosage is crucial for proper stem cell homeostasis and prevention of cancer stem cell development. PMID:20804967

  7. Localization of a bacterial group II intron-encoded protein in human cells.

    PubMed

    Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

    2015-01-01

    Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

  8. Group I intron located in PR protein homologue gene in Youngia japonica.

    PubMed

    Nishida, H; Ogura, A; Yokota, A; Yamaguchi, I; Sugiyama, J

    2000-03-01

    A Youngia japonica strain had a group I intron that was suggested to have been transferred from Protomyces inouyei, a pathogenic fungus of Y. japonica. It was located in the miraculin homologue coding gene by reverse complementation. The deduced amino acid sequence of this miraculin homologue of Y. japonica was similar to the amino acid sequences of tobacco and tomato pathogenesis-related proteins. PMID:10803963

  9. Monoclonal antibodies directed against VP7 protein of human group B rotavirus.

    PubMed

    Deng, Xiaojie; Xiong, Guomei; Cong, Wenjuan; Liu, Zhonglai; Qi, Chao; Yang, Jihong

    2014-02-01

    The aim of this study was to prepare and identify a monoclonal antibody that binds the viral proteins 7 (VP7 protein) of human group B rotavirus (GBRV) and to describe its immunologic characterization. Human group B rotavirus vp7 gene was successfully ligated into pGEX-KG vector and transformed into Escherichia coli TOP10 cells. The glutathione S-transferases (GST)-fusion protein GST-VP7 was induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and immediately purified to immunize BALB/c mice. Splenocytes were then prepared from the immunized mouse and fused with SP2/0 myeloma cell line. In the end we obtained one positive hybridoma cell line stably secreting monoclonal antibody against GST-VP7 protein by indirect enzyme-linked immunosorbent assay (ELISA) and limiting dilution. The production of the monoclonal antibody against GBRV will benefit the further study of GBRV's structures and functions and also lay a solid foundation for the research of disease prevention, clinical diagnosis, and treatment. PMID:24555935

  10. R4 regulators of G protein signaling (RGS) identify an ancient MHC-linked synteny group

    PubMed Central

    Suurväli, Jaanus; Robert, Jacques; Boudinot, Pierre; Boudinot, Sirje Rüütel

    2012-01-01

    Regulators of G Protein Signaling (RGS) are key regulators of G protein signaling. RGS proteins of the R4 RGS group are composed of a mere RGS domain and are mainly involved in immune response modulation. In both human and mouse, most genes encoding the R4 RGS proteins are located in the same region of chromosome 1. We show here that the RGS1/RGS16 neighborhood constitutes a synteny group well conserved across tetrapods, and closely linked to the MHC paralogon of chromosome 1. Genes located in the RGS1/RGS16 region have paralogs close to the MHC on chromosome 6 or close to the other MHC paralogons. In amphioxus, a cephalochordate, these genes possess orthologs that are located in the same scaffolds as a number of markers defining the proto-MHC in this species (Abi-Rached et al. 2002). We therefore propose that the RGS1/RGS16 region provides useful markers to investigate the origins and the evolution of the MHC. In addition, we show that some genes of the region appear to have immune functions not only in human, but also in Xenopus. PMID:23129146

  11. Localizing chemical groups while imaging single native proteins by high-resolution atomic force microscopy.

    PubMed

    Pfreundschuh, Moritz; Alsteens, David; Hilbert, Manuel; Steinmetz, Michel O; Müller, Daniel J

    2014-05-14

    Simultaneous high-resolution imaging and localization of chemical interaction sites on single native proteins is a pertinent biophysical, biochemical, and nanotechnological challenge. Such structural mapping and characterization of binding sites is of importance in understanding how proteins interact with their environment and in manipulating such interactions in a plethora of biotechnological applications. Thus far, this challenge remains to be tackled. Here, we introduce force-distance curve-based atomic force microscopy (FD-based AFM) for the high-resolution imaging of SAS-6, a protein that self-assembles into cartwheel-like structures. Using functionalized AFM tips bearing Ni(2+)-N-nitrilotriacetate groups, we locate specific interaction sites on SAS-6 at nanometer resolution and quantify the binding strength of the Ni(2+)-NTA groups to histidine residues. The FD-based AFM approach can readily be applied to image any other native protein and to locate and structurally map histidine residues. Moreover, the surface chemistry used to functionalize the AFM tip can be modified to map other chemical interaction sites. PMID:24766578

  12. The controversial role of the Polycomb group proteins in transcription and cancer: how much do we not understand Polycomb proteins?

    PubMed

    Scelfo, Andrea; Piunti, Andrea; Pasini, Diego

    2015-05-01

    Polycomb group proteins (PcGs) are a large protein family that includes diverse biochemical features assembled together in two large multiprotein complexes. These complexes maintain gene transcriptional repression in a cell type specific manner by modifying the surrounding chromatin to control development, differentiation and cell proliferation. PcGs are also involved in several diseases. PcGs are often directly or indirectly implicated in cancer development for which they have been proposed as potential targets for cancer therapeutic strategies. However, in the last few years a series of discoveries about the basic properties of PcGs and the identification of specific genetic alterations affecting specific Polycomb proteins in different tumours have converged to challenge old dogmas about PcG biological and molecular functions. In this review, we analyse these new data in the context of the old knowledge, highlighting the controversies and providing new models of interpretation and ideas that will perhaps bring some order among apparently contradicting observations. PMID:25315766

  13. Quinone-induced protein modifications: Kinetic preference for reaction of 1,2-benzoquinones with thiol groups in proteins.

    PubMed

    Li, Yuting; Jongberg, Sisse; Andersen, Mogens L; Davies, Michael J; Lund, Marianne N

    2016-08-01

    Oxidation of polyphenols to quinones serves as an antioxidative mechanism, but the resulting quinones may induce damage to proteins as they react through a Michael addition with nucleophilic groups, such as thiols and amines to give protein adducts. In this study, rate constants for the reaction of 4-methylbenzoquinone (4MBQ) with proteins, thiol and amine compounds were determined under pseudo first-order conditions by UV-vis stopped-flow spectrophotometry. The chemical structures of the adducts were identified by LC-ESI-MS/MS. Proteins with free thiols were rapidly modified by 4MBQ with apparent second order rate constants, k2 of (3.1±0.2)×10(4)M(-1)s(-1) for bovine serum albumin (BSA) and (4.8±0.2)×10(3)M(-1)s(-1) for human serum albumin at pH 7.0. These values are at least 12-fold greater than that for α-lactalbumin (4.0±0.2)×10(2)M(-1)s(-1), which does not contain any free thiols. Reaction of Cys-34 of BSA with N-ethylmaleimide reduced the thiol concentration by ~59%, which resulted in a decrease in k2 by a similar percentage, consistent with rapid adduction at Cys-34. Reaction of 4MBQ with amines (Gly, Nα-acetyl-l-Lys, Nε-acetyl-l-Lys and l-Lys) and the guanidine group of Nα-acetyl-l-Arg was at least 5×10(5) slower than with low-molecular-mass thiols (l-Cys, Nα-acetyl-l-Cys, glutathione). The thiol-quinone interactions formed colorless thiol-phenol products via an intermediate adduct, while the amine-quinone interactions generated colored amine-quinone products that require oxygen involvement. These data provide strong evidence for rapid modification of protein thiols by quinone species which may be of considerable significance for biological and food systems. PMID:27212016

  14. The influence of glutamatergic receptor antagonists on biochemical and ultrastructural changes in myelin membranes of rats subjected to experimental autoimmune encephalomyelitis.

    PubMed

    Dąbrowska-Bouta, Beata; Strużyńska, Lidia; Chalimoniuk, Małgorzata; Frontczak-Baniewicz, Małgorzata; Sulkowski, Grzegorz

    2015-01-01

    Elevated extracellular glutamate in the synaptic cleft causes overactivation of glutamate receptors and kills neurons by an excitotoxic mechanism. Recent studies have shown that glutamate can also lead to toxic injury of white matter oligodendrocytes in myelin sheaths and consequently to axon demyelination. The present study was performed using the rodent model of multiple sclerosis known as experimental autoimmune encephalomyelitis (EAE). The aim of the study was to test the effects of the glutamatergic receptor antagonists amantadine and memantine (antagonists of NMDA receptors), LY 367384 (an antagonist of mGluR1), and MPEP (an mGluR5 antagonist) on the development of neurological symptoms in immunized animals, morphological changes in cerebral myelin, and expression of mRNA of the principal myelin proteins PLP, MBP, MOG, MAG, and CNPase. Pharmacological inhibition of NMDA receptors by amantadine and memantine was found to suppress neurological symptoms in EAE rats, whereas antagonists of the group I metabotropic glutamate receptors (mGluRs G I) did not function positively. In the symptomatic phase of the disease we observed destruction of myelin sheaths via electron microscopy and decreased levels of mRNA for all of the principal myelin proteins. The results reveal that glutamate receptor antagonists have a positive effect on the expression of mRNA MBP and glycoproteins MAG and MOG but not on myelin ultrastructure. PMID:26785366

  15. Xeroderma Pigmentosum Group A Protein Loads as a Separate Factor onto DNA Lesions

    PubMed Central

    Rademakers, Suzanne; Volker, Marcel; Hoogstraten, Deborah; Nigg, Alex L.; Moné, Martijn J.; van Zeeland, Albert A.; Hoeijmakers, Jan H. J.; Houtsmuller, Adriaan B.; Vermeulen, Wim

    2003-01-01

    Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient (∼5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair. PMID:12897146

  16. High Mobility Group B Proteins, Their Partners, and Other Redox Sensors in Ovarian and Prostate Cancer

    PubMed Central

    Barreiro-Alonso, Aida; Lamas-Maceiras, Mónica; Rodríguez-Belmonte, Esther; Vizoso-Vázquez, Ángel; Quindós, María; Cerdán, M. Esperanza

    2016-01-01

    Cancer cells try to avoid the overproduction of reactive oxygen species by metabolic rearrangements. These cells also develop specific strategies to increase ROS resistance and to express the enzymatic activities necessary for ROS detoxification. Oxidative stress produces DNA damage and also induces responses, which could help the cell to restore the initial equilibrium. But if this is not possible, oxidative stress finally activates signals that will lead to cell death. High mobility group B (HMGB) proteins have been previously related to the onset and progressions of cancers of different origins. The protein HMGB1 behaves as a redox sensor and its structural changes, which are conditioned by the oxidative environment, are associated with different functions of the protein. This review describes recent advances in the role of human HMGB proteins and other proteins interacting with them, in cancerous processes related to oxidative stress, with special reference to ovarian and prostate cancer. Their participation in the molecular mechanisms of resistance to cisplatin, a drug commonly used in chemotherapy, is also revised. PMID:26682011

  17. Group A Streptococcus Adheres to Pharyngeal Epithelial Cells with Salivary Proline-rich Proteins via GrpE Chaperone Protein*

    PubMed Central

    Murakami, Jumpei; Terao, Yutaka; Morisaki, Ichijiro; Hamada, Shigeyuki; Kawabata, Shigetada

    2012-01-01

    Group A Streptococcus pyogenes (GAS) is an important human pathogen that frequently causes pharyngitis. GAS organisms can adhere to and invade pharyngeal epithelial cells, which are overlaid by salivary components. However, the role of salivary components in GAS adhesion to pharyngeal cells has not been reported precisely. We collected human saliva and purified various salivary components, including proline-rich protein (PRP), statherin, and amylase, and performed invasion assays. The GAS-HEp-2 association ratio (invasion/adhesion ratio) and invasion ratio of GAS were increased significantly with whole human saliva and PRP, while the anti-PRP antibody inhibited the latter. GAS strain NY-5, which lacks M and F proteins on the cell surface, was promoted to cohere with HEp-2 cells by whole human saliva and PRP. The 28-kDa protein of GAS bound to PRP and was identified as GrpE, a chaperone protein, whereas the N-terminal of GrpE was found to bind to PRP. A GrpE-deficient mutant of GAS strain B514Sm, TR-45, exhibited a reduced ability to adhere to and invade HEp-2 cells. Microscopic observations showed the GrpE was mainly expressed on the surface of the cell division site of GAS. Furthermore, GrpE-deficient mutants of GAS and Streptococcus pneumoniae showed an elongated morphology as compared with the wild type. Taken together, this is the first study to show an interaction between salivary PRP and GAS GrpE, which plays an important role in GAS infection on the pharynx, whereas the expression of GrpE on the surface of GAS helps to maintain morphology. PMID:22566698

  18. Entropy Loss of Hydroxyl Groups of Balanol upon Binding to Protein Kinase A

    NASA Astrophysics Data System (ADS)

    Gidofalvi, Gergely; Wong, Chung F.; McCammon, J. Andrew

    2002-09-01

    This article describes a short project for an undergraduate to learn several techniques for computer-aided drug design. The project involves estimating the loss of the rotational entropy of the hydroxyl groups of balanol upon its binding to the enzyme protein kinase A (PKA), as the entropy loss can significantly influence PKA balanol binding affinity. This work employs semiempirical quantum mechanical techniques for estimating the potential energy curves for the rotation of the hydroxyl groups of balanol in vacuum and in PKA, and solves the Poisson equation to correct the potential energy curves for hydration effects. Statistical mechanical principles are then applied to estimate the desired entropy loss from the potential energy curves. The analysis examines the influence of hydration effects on the rotational preference of the hydroxyl groups and the significance of the rotational entropy in determining binding affinity.

  19. Identification of a group of Haemophilus influenzae penicillin-binding proteins that may have complementary physiological roles

    SciTech Connect

    Malouin, F.; Parr, T.R. Jr.; Bryan, L.E. )

    1990-02-01

    (35S)penicillin bound to different Haemophilus influenzae proteins in assays performed at 20, 37, or 42{degrees}C. Penicillin-binding proteins 3a, 3b, 4, and 4' formed a group characterized by their affinity for moxalactam, cefotaxime, and piperacillin. Penicillin-binding protein 4' showed specific properties that may reflect its complementary role in septation.

  20. Conformational relaxation and water penetration coupled to ionization of internal groups in proteins

    PubMed Central

    Damjanović, Ana; Brooks, Bernard R.; Bertrand García-Moreno, E

    2012-01-01

    Molecular dynamics simulations were used to examine the effects of ionization of internal groups on the structures of eighteen variants of staphylococcal nuclease (SNase) with internal Lys, Asp, or Glu. In most cases the RMSD values of internal ionizable side chains were larger when the ionizable moieties were charged than when they were neutral. Calculations of solvent-accessible surface area showed that the internal ionizable side chains were buried in the protein interior when they were neutral, and moved towards crevices and the protein-water interface when they were charged. The only exceptions are Lys-36, Lys-62, Lys-92 and Lys-103, which remained buried even after charging. With the exception of Lys-38, the number of internal water molecules surrounding the ionizable group increased upon charging: the average number of water oxygen atoms within the first hydration shell increased by 1.7 for Lys residues, by 5.2 for Asp residues, and by 3.2 for Glu residues. The polarity of the micro environment of the ionizable group also increased when the groups were charged: the average number of polar atoms of any kind within the first hydration shell increased by 2.7 for Lys residues, by 4.8 for Asp residues, and by 4.0 for Glu residues. An unexpected linear relationship was observed between the absolute value of the shifts in pKa values measured experimentally, and structural relaxation as described in terms of the net difference in the polarity of the micro environment of the charged and neutral forms of the ionizable groups, and of the RMSD values of the charged side chains. The effects of ionization of internal groups on the conformation of the backbone were noticeable but mostly small and localized to the area immediately next to the internal ionizable moiety. Some variants did exhibit local unfolding. PMID:21428436

  1. High mobility group nucleosome-binding family proteins promote astrocyte differentiation of neural precursor cells.

    PubMed

    Nagao, Motoshi; Lanjakornsiripan, Darin; Itoh, Yasuhiro; Kishi, Yusuke; Ogata, Toru; Gotoh, Yukiko

    2014-11-01

    Astrocytes are the most abundant cell type in the mammalian brain and are important for the functions of the central nervous system. Although previous studies have shown that the STAT signaling pathway or its regulators promote the generation of astrocytes from multipotent neural precursor cells (NPCs) in the developing mammalian brain, the molecular mechanisms that regulate the astrocytic fate decision have still remained largely unclear. Here, we show that the high mobility group nucleosome-binding (HMGN) family proteins, HMGN1, 2, and 3, promote astrocyte differentiation of NPCs during brain development. HMGN proteins were expressed in NPCs, Sox9(+) glial progenitors, and GFAP(+) astrocytes in perinatal and adult brains. Forced expression of either HMGN1, 2, or 3 in NPCs in cultures or in the late embryonic neocortex increased the generation of astrocytes at the expense of neurons. Conversely, knockdown of either HMGN1, 2, or 3 in NPCs suppressed astrocyte differentiation and promoted neuronal differentiation. Importantly, overexpression of HMGN proteins did not induce the phosphorylation of STAT3 or activate STAT reporter genes. In addition, HMGN family proteins did not enhance DNA demethylation and acetylation of histone H3 around the STAT-binding site of the gfap promoter. Moreover, knockdown of HMGN family proteins significantly reduced astrocyte differentiation induced by gliogenic signal ciliary neurotrophic factor, which activates the JAK-STAT pathway. Therefore, we propose that HMGN family proteins are novel chromatin regulatory factors that control astrocyte fate decision/differentiation in parallel with or downstream of the JAK-STAT pathway through modulation of the responsiveness to gliogenic signals. PMID:25069414

  2. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

    SciTech Connect

    Price, E.M.; Freisheim, J.H.

    1987-07-28

    A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a K/sub t/ of 506 +/- 79 nM and a V/sub max/ of 17.9 +/- 4.2 pmol min/sup -1/ (mg of total cellular protein)/sup -1/. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate. The parent compounds of the iodinated photoprobe inhibit (/sup 3/H)methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a K/sub i/ of 66 +/- 21 nM. UV irradiation, at 4 /sup 0/C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the K/sub t/ for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. Evidence that, in the absence of irradiation and at 37/sup 0/C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (M/sub r/ 38K and 21K) derived from the cell homogenate supernatant.

  3. A short PPR protein required for the splicing of specific group II introns in angiosperm chloroplasts

    PubMed Central

    Khrouchtchova, Anastassia; Monde, Rita-Ann; Barkan, Alice

    2012-01-01

    A maize gene designated thylakoid assembly 8 (tha8) emerged from a screen for nuclear mutations that cause defects in the biogenesis of chloroplast thylakoid membranes. The tha8 gene encodes an unusual member of the pentatricopeptide repeat (PPR) family, a family of helical repeat proteins that participate in various aspects of organellar RNA metabolism. THA8 localizes to chloroplasts, where it associates specifically with the ycf3-2 and trnA group II introns. The splicing of ycf3-2 is eliminated in tha8 mutants, and trnA splicing is strongly compromised. Reverse-genetic analysis of the tha8 ortholog in Arabidopsis thaliana showed that these molecular functions are conserved, although null alleles are embryo lethal in Arabidopsis and seedling lethal in maize. Whereas most PPR proteins have more than 10 PPR motifs, THA8 belongs to a subfamily of plant PPR proteins with only four PPR motifs and little else. THA8 is the first member of this subfamily with a defined molecular function, and illustrates that even small PPR proteins have the potential to mediate specific intermolecular interactions in vivo. PMID:22495966

  4. SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair

    PubMed Central

    Akita, Masaki; Tak, Yon-Soo; Shimura, Tsutomu; Matsumoto, Syota; Okuda-Shimizu, Yuki; Shimizu, Yuichiro; Nishi, Ryotaro; Saitoh, Hisato; Iwai, Shigenori; Mori, Toshio; Ikura, Tsuyoshi; Sakai, Wataru; Hanaoka, Fumio; Sugasawa, Kaoru

    2015-01-01

    The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER. PMID:26042670

  5. Group 3 LEA protein model peptides protect enzymes against desiccation stress.

    PubMed

    Furuki, Takao; Sakurai, Minoru

    2016-09-01

    We tested whether model peptides for group 3 late embryogenesis abundant (G3LEA) proteins, which we developed previously, are capable of maintaining the catalytic activities of enzymes dried in their presence. Three different peptides were compared: 1) PvLEA-22, which consists of two tandem repeats of the 11-mer motif found in G3LEA proteins from an African sleeping chironomid; 2) PvLEA-44, which is made of four tandem repeats of the same 11-mer motif; and 3) a peptide whose amino acid composition is the same as that of PvLEA-22, but whose sequence is scrambled. We selected two enzymes, lactate dehydrogenase (LDH) and β-d-galactosidase (BDG), as targets because they have different isoelectric point (pI) values, in the alkaline and acidic range, respectively. While these enzymes were almost inactivated when dried alone, their catalytic activity was preserved at ≥70% of native levels in the presence of any of the above three peptides. This degree of protection is comparable to that conferred by several full-length G3LEA proteins, as reported previously for LDH. Interestingly, the protective activity of the peptides was enhanced slightly when they were mixed with trehalose, especially when the molar content of the peptides was low. On the basis of these results, the G3LEA model peptides show promise as protectants for the dry preservation of enzymes/proteins with a wide range of pI values. PMID:27131872

  6. Structure and Assembly of Group B Streptococcus Pilus 2b Backbone Protein

    PubMed Central

    Cozzi, Roberta; Malito, Enrico; Lazzarin, Maddalena; Nuccitelli, Annalisa; Castagnetti, Andrea; Bottomley, Matthew J.; Margarit, Immaculada; Maione, Domenico; Rinaudo, C. Daniela

    2015-01-01

    Group B Streptococcus (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization. PMID:25942637

  7. TATA-Binding Protein Mutants That Are Lethal in the Absence of the Nhp6 High-Mobility-Group Protein

    PubMed Central

    Eriksson, Peter; Biswas, Debabrata; Yu, Yaxin; Stewart, James M.; Stillman, David J.

    2004-01-01

    The Saccharomyces cerevisiae Nhp6 protein is related to the high-mobility-group B family of architectural DNA-binding proteins that bind DNA nonspecifically but bend DNA sharply. Nhp6 is involved in transcriptional activation by both RNA polymerase II (Pol II) and Pol III. Our previous genetic studies have implicated Nhp6 in facilitating TATA-binding protein (TBP) binding to some Pol II promoters in vivo, and we have used a novel genetic screen to isolate 32 new mutations in TBP that are viable in wild-type cells but lethal in the absence of Nhp6. The TBP mutations that are lethal in the absence of Nhp6 cluster in three regions: on the upper surface of TBP that may have a regulatory role, near residues that contact Spt3, or near residues known to contact either TFIIA or Brf1 (in TFIIIB). The latter set of mutations suggests that Nhp6 becomes essential when a TBP mutant compromises its ability to interact with either TFIIA or Brf1. Importantly, the synthetic lethality for some of the TBP mutations is suppressed by a multicopy plasmid with SNR6 or by an spt3 mutation. It has been previously shown that nhp6ab mutants are defective in expressing SNR6, a Pol III-transcribed gene encoding the U6 splicing RNA. Chromatin immunoprecipitation experiments show that TBP binding to SNR6 is reduced in an nhp6ab mutant. Nhp6 interacts with Spt16/Pob3, the yeast equivalent of the FACT elongation complex, consistent with nhp6ab cells being extremely sensitive to 6-azauracil (6-AU). However, this 6-AU sensitivity can be suppressed by multicopy SNR6 or BRF1. Additionally, strains with SNR6 promoter mutations are sensitive to 6-AU, suggesting that decreased SNR6 RNA levels contribute to 6-AU sensitivity. These results challenge the widely held belief that 6-AU sensitivity results from a defect in transcriptional elongation. PMID:15226442

  8. Diffusion-enhanced lanthanide energy transfer studies of protein prosthetic groups

    SciTech Connect

    Meares, C.F.; Yeh, S.M.; Rice, L.S.

    1980-10-01

    A long-lived luminescent solute in aqueous solution (e.g., /sup 5/D/sub 4/ terbium, tau approx. =10/sup -3/s) can donate its excitation energy to a chromophore such as a protein prosthetic group by, e.g., the radiationless dipolar mechanism of Foerster. However, in contrast to the usual energy-transfer experiment, a donor with a 10/sup -3/s lifetime can diffuse extensively through the solution and, in a time scale short compared to its excited lifetime, sample all permitted locations with respect to chromophoric acceptors. As recently indicated by Thomas et al. energy transfer in this rapid-diffusion limit can permit direct measurement of the allowed distance of closest approach of small solute molecules to chromophores which may be buried within proteins or membranes.

  9. Nuclear Trafficking of the Rabies Virus Interferon Antagonist P-Protein Is Regulated by an Importin-Binding Nuclear Localization Sequence in the C-Terminal Domain

    PubMed Central

    Rowe, Caitlin L.; Wagstaff, Kylie M.; Oksayan, Sibil; Glover, Dominic J.

    2016-01-01

    Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein

  10. Nuclear Trafficking of the Rabies Virus Interferon Antagonist P-Protein Is Regulated by an Importin-Binding Nuclear Localization Sequence in the C-Terminal Domain.

    PubMed

    Rowe, Caitlin L; Wagstaff, Kylie M; Oksayan, Sibil; Glover, Dominic J; Jans, David A; Moseley, Gregory W

    2016-01-01

    Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein

  11. Multiple Targeting Approaches on Histamine H3 Receptor Antagonists

    PubMed Central

    Khanfar, Mohammad A.; Affini, Anna; Lutsenko, Kiril; Nikolic, Katarina; Butini, Stefania; Stark, Holger

    2016-01-01

    With the very recent market approval of pitolisant (Wakix®), the interest in clinical applications of novel multifunctional histamine H3 receptor antagonists has clearly increased. Since histamine H3 receptor antagonists in clinical development have been tested for a variety of different indications, the combination of pharmacological properties in one molecule for improved pharmacological effects and reduced unwanted side-effects is rationally based on the increasing knowledge on the complex neurotransmitter regulations. The polypharmacological approaches on histamine H3 receptor antagonists on different G-protein coupled receptors, transporters, enzymes as well as on NO-signaling mechanism are described, supported with some lead structures. PMID:27303254

  12. Multiple Targeting Approaches on Histamine H3 Receptor Antagonists.

    PubMed

    Khanfar, Mohammad A; Affini, Anna; Lutsenko, Kiril; Nikolic, Katarina; Butini, Stefania; Stark, Holger

    2016-01-01

    With the very recent market approval of pitolisant (Wakix®), the interest in clinical applications of novel multifunctional histamine H3 receptor antagonists has clearly increased. Since histamine H3 receptor antagonists in clinical development have been tested for a variety of different indications, the combination of pharmacological properties in one molecule for improved pharmacological effects and reduced unwanted side-effects is rationally based on the increasing knowledge on the complex neurotransmitter regulations. The polypharmacological approaches on histamine H3 receptor antagonists on different G-protein coupled receptors, transporters, enzymes as well as on NO-signaling mechanism are described, supported with some lead structures. PMID:27303254

  13. Corticospinal control of antagonistic muscles in the cat.

    PubMed

    Ethier, Christian; Brizzi, Laurent; Giguère, Dominic; Capaday, Charles

    2007-09-01

    We recently suggested that movement-related inter-joint muscle synergies are recruited by selected excitation and selected release from inhibition of cortical points. Here we asked whether a similar cortical mechanism operates in the functional linking of antagonistic muscles. To this end experiments were done on ketamine-anesthetized cats. Intracortical microstimulation (ICMS) and intramuscular electromyographic recordings were used to find and characterize wrist, elbow and shoulder antagonistic motor cortical points. Simultaneous ICMS applied at two cortical points, each evoking activity in one of a pair of antagonistic muscles, produced co-contraction of antagonistic muscle pairs. However, we found an obvious asymmetry in the strength of reciprocal inhibition; it was always significantly stronger on physiological extensors than flexors. Following intravenous injection of a single bolus of strychnine, a cortical point at which only a physiological flexor was previously activated also elicited simultaneous activation of its antagonist. This demonstrates that antagonistic corticospinal neurons are closely grouped, or intermingled. To test whether releasing a cortical point from inhibition allows it to be functionally linked with an antagonistic cortical point, one of three GABA(A) receptor antagonists, bicuculline, gabazine or picrotoxin, was injected iontophoretically at one cortical point while stimulation was applied to an antagonistic cortical point. This coupling always resulted in co-contraction of the represented antagonistic muscles. Thus, antagonistic motor cortical points are linked by excitatory intracortical connections held in check by local GABAergic inhibition, with reciprocal inhibition occurring at the spinal level. Importantly, the asymmetry of cortically mediated reciprocal inhibition would appear significantly to bias muscle maps obtained by ICMS in favor of physiological flexors. PMID:17880397

  14. Decreased organ failure in patients with severe SIRS and septic shock treated with the platelet-activating factor antagonist TCV-309: a prospective, multicenter, double-blind, randomized phase II trial. TCV-309 Septic Shock Study Group.

    PubMed

    Poeze, M; Froon, A H; Ramsay, G; Buurman, W A; Greve, J W

    2000-10-01

    Sepsis and organ failure remain the main cause of death on the ICU. Sepsis is characterized by a severe inflammatory response, in which platelet-activating factor (PAF) is considered to play an important role. This study investigated whether treatment with the PAF-antagonist TCV-309 reduces morbidity and mortality in patients with septic shock. The study was conducted as a double-blind, randomized, placebo controlled multicenter study. The included patients had to fulfill the SIRS criteria with a clinical suspicion of infection, an admission APACHE II score greater than 15, and shock, defined as a mean arterial pressure <70 mmHg and/or a decrease > or =40 mmHg despite adequate fluid resuscitation. Patients received 1.0 mg/kg TCV-309 or placebo, twice daily, intravenously during 14 days. The prospectively set goals were MOF score, recovery from shock, mortality, and assessment of the safety of the medication. A total of 98 patients were included of which 97 were analyzed on an intention-to-treat basis. The overall survival at day 56 of TCV-309 treated patients was similar compared to placebo treated patients (51.0% vs. 41.7%, P = 0.47). In contrast, the mean percentage of failed organs per patient present after 14 days in the TCV-309 treated patients was significantly lower compared to the placebo treated patients (11.9% vs. 25.1%, P = 0.04), leading to a reduced need for vasopressors, dialysis, and ventilatory support. Furthermore, the mean APACHE-II score during treatment with TCV-309 was significantly lower and the number of patients recovered from shock after day 14 was significantly higher in the TCV-309 treated patient group (2/32 vs. 9/29, P = 0.01). The number of adverse events was not significantly different between the TCV-309 and placebo treated patients. TCV-309 did not change overall mortality of septic shock, however a substantial reduction in organ dysfunction and morbidity, frequently associated with septic shock was achieved, without significant

  15. Group 1 LEA proteins contribute to the desiccation and freeze tolerance of Artemia franciscana embryos during diapause.

    PubMed

    Toxopeus, Jantina; Warner, Alden H; MacRae, Thomas H

    2014-11-01

    Water loss either by desiccation or freezing causes multiple forms of cellular damage. The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis-life without water-during diapause. To better understand how cysts survive reduced hydration, group 1 late embryogenesis abundant (LEA) proteins, hydrophilic unstructured proteins that accumulate in the stress-tolerant cysts of A. franciscana, were knocked down using RNA interference (RNAi). Embryos lacking group 1 LEA proteins showed significantly lower survival than control embryos after desiccation and freezing, or freezing alone, demonstrating a role for group 1 LEA proteins in A. franciscana tolerance of low water conditions. In contrast, regardless of group 1 LEA protein presence, cysts responded similarly to hydrogen peroxide (H2O2) exposure, indicating little to no function for these proteins in diapause termination. This is the first in vivo study of group 1 LEA proteins in an animal and it contributes to the fundamental understanding of these proteins. Knowing how LEA proteins protect A. franciscana cysts from desiccation and freezing may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications. PMID:24846336

  16. “Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis*

    PubMed Central

    Ho, Chia Chi M.; Guo, Nan; Sockolosky, Jonathan T.; Ring, Aaron M.; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L.; Garcia, K. Christopher

    2015-01-01

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don't-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy

  17. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani.

    PubMed

    McNeil, Bonnie A; Simon, Dawn M; Zimmerly, Steven

    2014-02-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5' splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5' exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns. PMID:24214997

  18. Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist

    PubMed Central

    Go, Yun Young; Li, Yanhua; Chen, Zhenhai; Han, Mingyuan; Yoo, Dongwan; Fang, Ying; Balasuriya, Udeni B. R.

    2014-01-01

    The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-β was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-β mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-β promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response. PMID:24967365

  19. Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist

    SciTech Connect

    Chen, Z.; Lawson, S.; Sun, Z.; Zhou, X.; Guan, X.; Christopher-Hennings, J.; Nelson, E.A.; Fang, Y.

    2010-03-01

    The porcine reproductive and respiratory syndrome virus nsp1 is predicted to be auto-cleaved from the replicase polyprotein into nsp1alpha and nsp1beta subunits. In infected cells, we detected the actual existence of nsp1alpha and nsp1beta. Cleavage sites between nsp1alpha/nsp1beta and nsp1beta/nsp2 were identified by protein microsequencing analysis. Time course study showed that nsp1alpha and nsp1beta mainly localize into the cell nucleus after 10 h post infection. Further analysis revealed that both proteins dramatically inhibited IFN-beta expression. The nsp1beta was observed to significantly inhibit expression from an interferon-stimulated response element promoter after Sendai virus infection or interferon treatment. It was further determined to inhibit nuclear translocation of STAT1 in the JAK-STAT signaling pathway. These results demonstrated that nsp1beta has ability to inhibit both interferon synthesis and signaling, while nsp1alpha alone strongly inhibits interferon synthesis. These findings provide important insights into mechanisms of nsp1 in PRRSV pathogenesis and its impact in vaccine development.

  20. Extracellular High-Mobility Group Box 1 Protein (HMGB1) as a Mediator of Persistent Pain

    PubMed Central

    Agalave, Nilesh M; Svensson, Camilla I

    2014-01-01

    Although originally described as a highly conserved nuclear protein, high-mobility group box 1 protein (HMGB1) has emerged as a danger-associated molecular pattern molecule protein (DAMP) and is a mediator of innate and specific immune responses. HMGB1 is passively or actively released in response to infection, injury and cellular stress, providing chemotactic and cytokine-like functions in the extracellular environment, where it interacts with receptors such as receptor for advanced glycation end products (RAGE) and several Toll-like receptors (TLRs). Although HMGB1 was first revealed as a key mediator of sepsis, it also contributes to a number of other conditions and disease processes. Chronic pain arises as a direct consequence of injury, inflammation or diseases affecting the somatosensory system and can be devastating for the affected patients. Emerging data indicate that HMGB1 is also involved in the pathology of persistent pain. Here, we give an overview of HMGB1 as a proinflammatory mediator, focusing particularly on the role of HMGB1 in the induction and maintenance of hypersensitivity in experimental models of pain and discuss the therapeutic potential of targeting HMGB1 in conditions of chronic pain. PMID:25222915

  1. 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

  2. Structural Insight into Amino Group-carrier Protein-mediated Lysine Biosynthesis

    PubMed Central

    Yoshida, Ayako; Tomita, Takeo; Fujimura, Tsutomu; Nishiyama, Chiharu; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2015-01-01

    In the biosynthesis of lysine by Thermus thermophilus, the metabolite α-ketoglutarate is converted to the intermediate α-aminoadipate (AAA), which is protected by the 54-amino acid acidic protein LysW. In this study, we determined the crystal structure of LysZ from T. thermophilus (TtLysZ), an amino acid kinase that catalyzes the second step in the AAA to lysine conversion, which was in a complex with LysW at a resolution of 1.85 Å. A crystal analysis coupled with isothermal titration calorimetry of the TtLysZ mutants for TtLysW revealed tight interactions between LysZ and the globular and C-terminal extension domains of the LysW protein, which were mainly attributed to electrostatic forces. These results provided structural evidence for LysW acting as a protecting molecule for the α-amino group of AAA and also as a carrier protein to guarantee better recognition by biosynthetic enzymes for the efficient biosynthesis of lysine. PMID:25392000

  3. High-mobility Group Box-1 Protein Promotes Granulomatous Nephritis in Adenine-induced nephropathy

    PubMed Central

    Oyama, Yoko; Hashiguchi, Teruto; Taniguchi, Noboru; Tancharoen, Salunya; Uchimura, Tomonori; Biswas, Kamal K.; Kawahara, Ko-ichi; Nitanda, Takao; Umekita, Yoshihisa; Lotz, Martin; Maruyama, Ikuro

    2011-01-01

    Granulomatous nephritis can be triggered by diverse factors and results in kidney failure. However, despite accumulating data about granulomatous inflammation, pathogenetic mechanisms in nephritis remain unclear. The DNA-binding high-mobility group box-1 protein (HMGB1) initiates and propagates inflammation when released by activated macrophages, functions as an “alarm cytokine” signaling tissue damage. In this study, we demonstrated elevated HMGB1 expression in renal granulomas in rats with crystal-induced granulomatous nephritis caused by feeding an adenine-rich diet. HMGB1 levels were also raised in urine and serum, as well as monocyte chemoattractant protein-1 (MCP-1), a mediator of granulomatous inflammation. Injection of HMGB1 worsened renal function and upregulated MCP-1 in rats with crystal-induced granulomatous nephritis. HMGB1 also induced MCP-1 secretion through mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) pathways in rat renal tubular epithelial cells in vitro. Hmgb1+/− mice with crystal-induced nephritis displayed reduced MCP-1 expression in the kidneys and in urine and the number of macrophages in the kidneys was significantly decreased. We conclude that HMGB1 is a new mediator involved in crystal-induced nephritis that amplifies granulomatous inflammation in a cycle where MCP-1 attracts activated macrophages, resulting in excessive and sustained HMGB1 release. HMGB1 could be a novel target for inhibiting chronic granulomatous diseases. PMID:20231821

  4. Combining Ultracentrifugation and Peptide Termini Group-specific Immunoprecipitation for Multiplex Plasma Protein Analysis

    PubMed Central

    Volk, Sonja; Schreiber, Thomas D.; Eisen, David; Wiese, Calvin; Planatscher, Hannes; Pynn, Christopher J.; Stoll, Dieter; Templin, Markus F.; Joos, Thomas O.; Pötz, Oliver

    2012-01-01

    Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques. PMID:22527512

  5. Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins.

    PubMed

    Bankmann, M; Prakash, L; Prakash, S

    1992-02-01

    Xeroderma pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G. Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines. PMID:1741034

  6. The implication and potential applications of high-mobility group box 1 protein in breast cancer

    PubMed Central

    Sohun, Moonindranath

    2016-01-01

    High-mobility group box 1 protein (HMGB1) is a highly conserved, non-histone and ubiquitous chromosomal protein found enriched in active chromatin forming part of the high mobility group family of proteins and is encoded by the HMGB1 gene (13q12) in human beings. It has various intranuclear and extracellular functions. It plays an important role in the pathogenesis of many diseases including cancer. In 2012, there was approximately 1.67 million new breast cancer cases diagnosed which makes it the second most frequent cancer in the world after lung cancer (25% of all cancers) and the commonest cancer among women. Both pre-clinical and clinical studies have suggested that HMGB1 might be a useful target in the management of breast cancer. This review summarises the structure and functions of HMGB1 and its dual role in carcinogenesis both as a pro-tumorigenic and anti-tumorigenic factor. It also sums up evidence from in vitro and in vivo studies using breast cancer cell lines and samples which demonstrate its influence in radiotherapy, chemotherapy and hormonal therapy in breast cancer. It may have particular importance in HER2 positive and metastatic breast cancer. It might pave the way for new breast cancer treatments through development of novel drugs, use of microRNAs (miRNAs), targeting breast cancer stem cells (CSCs) and breast cancer immunotherapy. It may also play a role in determining breast cancer prognosis. Thus HMGB1 may open up novel avenues in breast cancer management. PMID:27386491

  7. The implication and potential applications of high-mobility group box 1 protein in breast cancer.

    PubMed

    Sohun, Moonindranath; Shen, Huiling

    2016-06-01

    High-mobility group box 1 protein (HMGB1) is a highly conserved, non-histone and ubiquitous chromosomal protein found enriched in active chromatin forming part of the high mobility group family of proteins and is encoded by the HMGB1 gene (13q12) in human beings. It has various intranuclear and extracellular functions. It plays an important role in the pathogenesis of many diseases including cancer. In 2012, there was approximately 1.67 million new breast cancer cases diagnosed which makes it the second most frequent cancer in the world after lung cancer (25% of all cancers) and the commonest cancer among women. Both pre-clinical and clinical studies have suggested that HMGB1 might be a useful target in the management of breast cancer. This review summarises the structure and functions of HMGB1 and its dual role in carcinogenesis both as a pro-tumorigenic and anti-tumorigenic factor. It also sums up evidence from in vitro and in vivo studies using breast cancer cell lines and samples which demonstrate its influence in radiotherapy, chemotherapy and hormonal therapy in breast cancer. It may have particular importance in HER2 positive and metastatic breast cancer. It might pave the way for new breast cancer treatments through development of novel drugs, use of microRNAs (miRNAs), targeting breast cancer stem cells (CSCs) and breast cancer immunotherapy. It may also play a role in determining breast cancer prognosis. Thus HMGB1 may open up novel avenues in breast cancer management. PMID:27386491

  8. Group A streptococcus expresses a trio of surface proteins containing protective epitopes.

    PubMed

    Niedermeyer, Shannon E; Penfound, Thomas A; Hohn, Claudia; Li, Yi; Homayouni, Ramin; Zhao, Jingnan; Dale, James B

    2014-10-01

    Group A streptococci (GAS) (Streptococcus pyogenes) are common causes of infections in humans for which there is no licensed vaccine. Decades of work has focused on the role of the surface M protein in eliciting type-specific protective immunity. Recent studies have identified additional surface proteins of GAS that contain opsonic epitopes. In the present study, we describe a serotype M65 GAS originally isolated during an epidemiologic study in Bamako, Mali, which simultaneously expressed M, M-related protein (Mrp), and streptococcal protective antigen (Spa) on the bacterial surface. The emm, mrp, and spa genes were sequenced from PCR amplicons derived from the M65 chromosome. Rabbit antisera raised against synthetic peptides copying the N-terminal regions of M, Mrp, and Spa were highly specific for each peptide, reacted with the surface of M65 GAS, and promoted bactericidal activity against the organism. A mixture of antisera against all three peptides was most effective in the bactericidal assays. Immunofluorescence microscopy revealed that the M, Mrp, and Spa antisera bound to the bacterial surface in the presence of human plasma proteins and resulted in the deposition of complement. Five additional spa genes were identified in the Mrp-positive GAS serotypes, and their sequences were determined. Our results indicate that there are multiple antigens on the surface of GAS that evoke antibodies that promote bacterial killing. A more complete understanding of the relative contributions of M, Mrp, and Spa in eliciting protective immunity may aid in the development of GAS vaccines with enhanced coverage and efficacy. PMID:25080552

  9. Contrasting evolutionary patterns of spore coat proteins in two Bacillus species groups are linked to a difference in cellular structure

    PubMed Central

    2013-01-01

    Background The Bacillus subtilis-group and the Bacillus cereus-group are two well-studied groups of species in the genus Bacillus. Bacteria in this genus can produce a highly resistant cell type, the spore, which is encased in a complex protective protein shell called the coat. Spores in the B. cereus-group contain an additional outer layer, the exosporium, which encircles the coat. The coat in B. subtilis spores possesses inner and outer layers. The aim of this study is to investigate whether differences in the spore structures influenced the divergence of the coat protein genes during the evolution of these two Bacillus species groups. Results We designed and implemented a computational framework to compare the evolutionary histories of coat proteins. We curated a list of B. subtilis coat proteins and identified their orthologs in 11 Bacillus species based on phylogenetic congruence. Phylogenetic profiles of these coat proteins show that they can be divided into conserved and labile ones. Coat proteins comprising the B. subtilis inner coat are significantly more conserved than those comprising the outer coat. We then performed genome-wide comparisons of the nonsynonymous/synonymous substitution rate ratio, dN/dS, and found contrasting patterns: Coat proteins have significantly higher dN/dS in the B. subtilis-group genomes, but not in the B. cereus-group genomes. We further corroborated this contrast by examining changes of dN/dS within gene trees, and found that some coat protein gene trees have significantly different dN/dS between the B subtilis-clade and the B. cereus-clade. Conclusions Coat proteins in the B. subtilis- and B. cereus-group species are under contrasting selective pressures. We speculate that the absence of the exosporium in the B. subtilis spore coat effectively lifted a structural constraint that has led to relaxed negative selection pressure on the outer coat. PMID:24283940

  10. Therapeutic approach to target mesothelioma cancer cells using the Wnt antagonist, secreted frizzled-related protein 4: Metabolic state of cancer cells.

    PubMed

    Perumal, Vanathi; Pohl, Sebastian; Keane, Kevin N; Arfuso, Frank; Newsholme, Philip; Fox, Simon; Dharmarajan, Arun

    2016-02-15

    Malignant mesothelioma (MM) is an aggressive cancer, characterized by rapid progression, along with late metastasis and poor patient prognosis. It is resistant to many forms of standard anti-cancer treatment. In this study, we determined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt pathway inhibitor, on cancer cell proliferation and metabolism using the JU77 mesothelioma cell line. Treatment with sFRP4 (250 pg/ml) resulted in a significant reduction of cell proliferation. The addition of the Wnt activator Wnt3a (250 pg/ml) or sFRP4 had no significant effect on ATP production and glucose utilisation in JU77 cells at both the 24 and 48 h time points examined. We also examined their effect on Akt and Glycogen synthase kinase-3 beta (GSK3β) phosphorylation, which are both important components of Wnt signalling and glucose metabolism. We found that protein phosphorylation of Akt and GSK3β varied over the 24h and 48 h time points, with constitutive phosphorylation of Akt at serine 473 (pAkt) decreasing to its most significant level when treated with Wnt3a+sFRP4 at the 24h time point. A significant reduction in the level of Cytochrome c oxidase was observed at the 48 h time point, when sFRP4 and Wnt3a were added in combination. We conclude that sFRP4 may function, in part, to reduce/alter cancer cell metabolism, which may lead to sensitisation of cancer cells to chemotherapeutics, or even cell death. PMID:26868304

  11. A protective surface protein from type V group B streptococci shares N-terminal sequence homology with the alpha C protein.

    PubMed Central

    Lachenauer, C S; Madoff, L C

    1996-01-01

    Infection by group B streptococci (GBS) is an important cause of bacterial disease in neonates, pregnant women, and nonpregnant adults. Historically, serotypes Ia, Ib, II, and III have been most prevalent among disease cases; recently, type V strains have emerged as important strains in the United States and elsewhere. In addition to type-specific capsular polysaccharides, many GBS strains possess surface proteins which demonstrate a laddering pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and resistance to trypsin digestion. These include the alpha C protein, the R proteins, and protein Rib. Some of these proteins elicit protective antibodies in animals. We demonstrate a trypsin-resistant laddering protein purified from a type V GBS strain by mutanolysin extraction and column chromatography. This protein contains a major 90-kDa band and a series of smaller bands spaced approximately 10 kDa apart on SDS-PAGE. Cross-reactivity of the type V protein with the alpha C protein and with R1 was demonstrated on Western blot (immunoblot). N-terminal sequence analysis of the protein revealed residue identity with 17 of 18 residues at corresponding positions on the alpha protein. Western blot of SDS extracts of 41 clinical type V isolates with rabbit antiserum to the protein demonstrated a homologous protein in 25 isolates (61%); two additional strains exhibited a heterologous pattern which was also demonstrated with 4G8, a monoclonal antibody directed to the alpha C protein repeat region. Rabbit antiserum raised to the type V protein conferred protection in neonatal mice against a type V strain bearing a homologous protein. These data support the hypothesis that there exists a family of trypsin-resistant, laddering GBS surface proteins which may play a role in immunity to GBS infection. PMID:8926097

  12. A protective surface protein from type V group B streptococci shares N-terminal sequence homology with the alpha C protein.

    PubMed

    Lachenauer, C S; Madoff, L C

    1996-10-01

    Infection by group B streptococci (GBS) is an important cause of bacterial disease in neonates, pregnant women, and nonpregnant adults. Historically, serotypes Ia, Ib, II, and III have been most prevalent among disease cases; recently, type V strains have emerged as important strains in the United States and elsewhere. In addition to type-specific capsular polysaccharides, many GBS strains possess surface proteins which demonstrate a laddering pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and resistance to trypsin digestion. These include the alpha C protein, the R proteins, and protein Rib. Some of these proteins elicit protective antibodies in animals. We demonstrate a trypsin-resistant laddering protein purified from a type V GBS strain by mutanolysin extraction and column chromatography. This protein contains a major 90-kDa band and a series of smaller bands spaced approximately 10 kDa apart on SDS-PAGE. Cross-reactivity of the type V protein with the alpha C protein and with R1 was demonstrated on Western blot (immunoblot). N-terminal sequence analysis of the protein revealed residue identity with 17 of 18 residues at corresponding positions on the alpha protein. Western blot of SDS extracts of 41 clinical type V isolates with rabbit antiserum to the protein demonstrated a homologous protein in 25 isolates (61%); two additional strains exhibited a heterologous pattern which was also demonstrated with 4G8, a monoclonal antibody directed to the alpha C protein repeat region. Rabbit antiserum raised to the type V protein conferred protection in neonatal mice against a type V strain bearing a homologous protein. These data support the hypothesis that there exists a family of trypsin-resistant, laddering GBS surface proteins which may play a role in immunity to GBS infection. PMID:8926097

  13. Calcium antagonists and their mode of action

    PubMed Central

    Nayler, Winifred G.; Dillon, J. S.

    1986-01-01

    1 The Ca2+ antagonists are a novel group of drugs useful in management of a variety of cardiac disorders. They differ from one another in terms of their chemistry, tissue specificity and selectivity. As a group, however, they share the common property of slowing Ca2+ entry through voltage-activated, ion-selective channels. Some of them exhibit other properties, including that of interfering with Na+ transport. At least one of them, diltiazem, has an intracellular action. 2 Specific high and low affinity binding sites have been identified for two of the major groups of Ca2+-antagonists, with the binding sites for verapamil and its derivatives being distinct from those which can be occupied by the dihydropyridines. The number (Bmax) and affinity (KD) of these binding sites changes under certain pathological conditions—including a reduction in ischaemia and in spontaneous hypertension, an increase in the latter, at present, only demonstrated for the dihydropyridine binding sites. 3 The sensitivity of a particular tissue to these drugs will depend upon a number of factors including the number of binding sites that are present, the contribution made by the Ca2+ entering through the voltage-activated channels to the functioning of the tissue, and properties which are peculiar to a particular type of Ca2+ antagonist, for example, whether, as in the case of verapamil, they exhibit use-dependence. PMID:3019374

  14. Calmodulin antagonists induce platelet apoptosis.

    PubMed

    Wang, Zhicheng; Li, Suping; Shi, Quanwei; Yan, Rong; Liu, Guanglei; Dai, Kesheng

    2010-04-01

    Calmodulin (CaM) antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. In platelets, CaM has been found to bind directly to the cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet functions. However, it is still unknown whether CaM antagonists induce platelet apoptosis. Here we show that CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), tamoxifen (TMX), and trifluoperazine (TFP) induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, and phosphatidylserine exposure. CaM antagonists did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP-, botrocetin-, and alpha-thrombin-induced platelet aggregation, platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonists. Furthermore, cytosolic Ca(2+) levels were obviously elevated by both W7 and TMX, and membrane-permeable Ca(2+) chelator BAPTA-AM significantly reduced apoptotic events in platelets induced by W7. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis. The elevation of the cytosolic Ca(2+) levels may be involved in the regulation of CaM antagonists-induced platelet apoptosis. PMID:20172594

  15. Localization and expression profile of Group I and II Activators of G-protein Signaling in the kidney.

    PubMed

    Lenarczyk, Marek; Pressly, Jeffrey D; Arnett, Joanna; Regner, Kevin R; Park, Frank

    2015-04-01

    Activators of G-protein Signaling (AGS) are a family of accessory proteins that were discovered as modulators of heterotrimeric G-protein subunits. The primary aim of the present study was to localize Group I and II AGS proteins and determine the renal expression profile using immunohistochemistry and quantitative RT-PCR, respectively, during normal and injured states of the kidney. Group I AGS1 was found to be predominantly localized to the proximal tubule, Group II AGS3 and AGS5 were exclusively localized to the distal tubular segments, and Group II AGS6 was ubiquitously expressed in every nephron segment of the rodent kidney. In rat kidneys following ischemia-reperfusion injury (IRI), Group I AGS1 mRNA was dramatically increased after 24 h by fivefold (P < 0.05), whereas Group II AGS3 and AGS4 mRNA was significantly decreased at the same time point (P < 0.05). No significant change in the transcript levels were detected at other time points for any of the AGS genes between control and IRI groups. In polycystic diseased kidneys, mRNA levels for AGS3, AGS4 and AGS6 was significantly increased (P < 0.05) by 75-80 % in PCK rat kidneys. The identification of Group I and II AGS mRNA and protein in the kidney may provide insight into the potential mechanism of action during normal and varying states of renal disease or injury. PMID:25533045

  16. DETECTION OF EXTRA-NUCLEAR HIGH MOBILITY GROUP BOX-1 PROTEIN IN A CANINE MODEL OF MYOCARDIAL INFARCTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The high mobility group box-1 protein (HMGB-1) is a well-characterized nuclear protein recently shown to be involved in endotoxin-induced inflammation and injury. Studies have linked HMGB-1 release to the production of pro-inflammatory cytokines; however, a role for HMGB-1 in other disorders involvi...

  17. Functionalized Congeners of P2Y1 Receptor Antagonists:

    SciTech Connect

    de Castro, Sonia; Maruoka, Hiroshi; Hong, Kunlun; Kilbey, II, S Michael; Costanzi, Stefano; Hechler, Béatrice; Gachet, Christian; Harden, T. Kendall; Jacobson, Kenneth A.

    2010-01-01

    The P2Y{sub 1} receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y{sub 1} receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N{sup 6}-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y{sub 1} receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of an intermediate amide group revealed high affinity of carboxylic congener 8 (K{sub i} 23 nM) and extended amine congener 15 (K{sub i} 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended {epsilon}-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y{sub 1} receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y{sub 1} receptor modeling and ligand docking. Attempted P2Y{sub 1} antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM dendrimer to

  18. Protein conformational exchange measured by 1H R1ρ relaxation dispersion of methyl groups.

    PubMed

    Weininger, Ulrich; Blissing, Annica T; Hennig, Janosch; Ahlner, Alexandra; Liu, Zhihong; Vogel, Hans J; Akke, Mikael; Lundström, Patrik

    2013-09-01

    Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for (1)H spins in methyl (13)CHD2 groups, which improves the characterization of fast exchange processes. The influence of (1)H-(1)H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R 1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different (1)H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any (1)H relaxation dispersion of its methyl groups at 10 or 25 °C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by (1)H and (13)C CPMG experiments. The present R 1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results. PMID:23904100

  19. Cutting edge: extracellular high mobility group box-1 protein is a proangiogenic cytokine.

    PubMed

    Mitola, Stefania; Belleri, Mirella; Urbinati, Chiara; Coltrini, Daniela; Sparatore, Bianca; Pedrazzi, Marco; Melloni, Edon; Presta, Marco

    2006-01-01

    The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus. PMID:16365390

  20. Correlates of Protection for M Protein-Based Vaccines against Group A Streptococcus

    PubMed Central

    Smeesters, Pierre R.; Frost, Hannah R. C.; Steer, Andrew C.

    2015-01-01

    Group A streptococcus (GAS) is known to cause a broad spectrum of illness, from pharyngitis and impetigo, to autoimmune sequelae such as rheumatic heart disease, and invasive diseases. It is a significant cause of infectious disease morbidity and mortality worldwide, but no efficacious vaccine is currently available. Progress in GAS vaccine development has been hindered by a number of obstacles, including a lack of standardization in immunoassays and the need to define human correlates of protection. In this review, we have examined the current immunoassays used in both GAS and other organisms, and explored the various challenges in their implementation in order to propose potential future directions to identify a correlate of protection and facilitate the development of M protein-based vaccines, which are currently the main GAS vaccine candidates. PMID:26101780

  1. Correlates of Protection for M Protein-Based Vaccines against Group A Streptococcus.

    PubMed

    Tsoi, Shu Ki; Smeesters, Pierre R; Frost, Hannah R C; Licciardi, Paul; Steer, Andrew C

    2015-01-01

    Group A streptococcus (GAS) is known to cause a broad spectrum of illness, from pharyngitis and impetigo, to autoimmune sequelae such as rheumatic heart disease, and invasive diseases. It is a significant cause of infectious disease morbidity and mortality worldwide, but no efficacious vaccine is currently available. Progress in GAS vaccine development has been hindered by a number of obstacles, including a lack of standardization in immunoassays and the need to define human correlates of protection. In this review, we have examined the current immunoassays used in both GAS and other organisms, and explored the various challenges in their implementation in order to propose potential future directions to identify a correlate of protection and facilitate the development of M protein-based vaccines, which are currently the main GAS vaccine candidates. PMID:26101780

  2. The Dimerization State of the Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2)

    PubMed Central

    Frost, Lorraine; Baez, Maria A. M.; Harrilal, Christopher; Garabedian, Alyssa; Fernandez-Lima, Francisco; Leng, Fenfei

    2015-01-01

    The mammalian high mobility group protein AT-hook 2 (HMGA2) is a chromosomal architectural transcription factor involved in cell transformation and oncogenesis. It consists of three positively charged “AT-hooks” and a negatively charged C-terminus. Sequence analyses, circular dichroism experiments, and gel-filtration studies showed that HMGA2, in the native state, does not have a defined secondary or tertiary structure. Surprisingly, using combined approaches of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemical cross-linking, analytical ultracentrifugation, fluorescence resonance energy transfer (FRET), and mass spectrometry, we discovered that HMGA2 is capable of self-associating into homodimers in aqueous buffer solution. Our results showed that electrostatic interactions between the positively charged “AT-hooks” and the negatively charged C-terminus greatly contribute to the homodimer formation. PMID:26114780

  3. A summary of the measured pK values of the ionizable groups in folded proteins.

    PubMed

    Grimsley, Gerald R; Scholtz, J Martin; Pace, C Nick

    2009-01-01

    We tabulated 541 measured pK values reported in the literature for the Asp, Glu, His, Cys, Tyr, and Lys side chains, and the C and N termini of 78 folded proteins. The majority of these values are for the Asp, Glu, and His side chains. The average pK values are Asp 3.5 +/- 1.2 (139); Glu 4.2 +/- 0.9 (153); His 6.6 +/- 1.0 (131); Cys 6.8 +/- 2.7 (25); Tyr 10.3 +/- 1.2 (20); Lys 10.5 +/- 1.1 (35); C-terminus 3.3 +/- 0.8 (22) and N-terminus 7.7 +/- 0.5 (16). We compare these results with the measured pK values of these groups in alanine pentapeptides, and comment on our overall findings. PMID:19177368

  4. Methyl group turnover on methyl-accepting chemotaxis proteins during chemotaxis by Bacillus subtilis

    SciTech Connect

    Thoelke, M.S.; Casper, J.M.; Ordal, G.W. )

    1990-02-05

    The addition of attractant to Bacillus subtilis briefly exposed to radioactive methionine causes an increase of labeling of the methyl-accepting chemotaxis proteins. The addition of attractant to cells radiolabeled for longer times shows no change in the extent of methylation. Therefore, the increase in labeling for the briefly labeled cells is due to an increased turnover of methyl groups caused by attractant. All amino acids gave enhanced turnover. This turnover lasted for a prolonged time, probably spanning the period of smooth swimming caused by the attractant addition. Repellent did not affect the turnover when added alone or simultaneously with attractant. Thus, for amino acid attractants, the turnover is probably the excitatory signal, which is seen to extend long into or throughout the adaptation period, not just at the start of it.

  5. A Nuclear Factor of High Mobility Group Box Protein in Toxoplasma gondii

    PubMed Central

    Wang, Hui; Lei, Tao; Liu, Jing; Li, Muzi; Nan, Huizhu; Liu, Qun

    2014-01-01

    High mobility group box 1 (HMGB1) is a nuclear factor that usually binds DNA and modulates gene expression in multicellular organisms. Three HMGB1 orthologs were predicted in the genome of Toxoplasma gondii, an obligate intracellular protozoan pathogen, termed TgHMGB1a, b and c. Phylogenetic and bioinformatic analyses indicated that these proteins all contain a single HMG box and which shared in three genotypes. We cloned TgHMGB1a, a 33.9 kDa protein that can stimulates macrophages to release TNF-α, and, we demonstrated that the TgHMGB1a binds distorted DNA structures such as cruciform DNA in electrophoretic mobility shift assays (EMSA). Immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites but translocated into the cytoplasm while the parasites release to extracellular. There were no significant phenotypic changes when the TgHMGB1a B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR analyses showed that the expression levels of many important genes, including virulence factors, increased when TgHMGB1a was overexpressed, but no significant changes were observed in TgHMGB1a B box-deficient parasites. Our findings demonstrated that TgHMGB1a is indeed a nuclear protein that maintains HMG box architectural functions and is a potential proinflammatory factor during the T.gondii infection. Further studies that clarify the functions of TgHMGB1s will increase our knowledge of transcriptional regulation and parasite virulence, and might provide new insight into host–parasite interactions for T. gondii infection. PMID:25369210

  6. Identification of a major, CsrRS-regulated secreted protein of Group A streptococcus.

    PubMed

    Heath, A; Miller, A; DiRita, V J; Engleberg, C N

    2001-08-01

    CsrR/CsrS (CovR/CovS) is a two-component regulator of extracellular virulence factors in Group A streptococcus, but the full range of regulated exoproteins is unknown. Since CsrR represses expression of regulated factors, culture supernates of wild-type and CsrR(-)mutant strains were compared by two-dimensional gel electrophoresis (2DGE) to identify regulated exoproteins. Supernates of DeltacsrRS(-)mutant, but not wild-type, bacteria contained an abundant 23 kDa protein. The N-terminal sequence of this spot corresponded to a putative open reading frame (ORF) in the streptococcal genome. In a mobility shift assay, phosphorylated CsrR bound to a PCR amplicon that included sequences upstream of this ORF. By primer extension analysis, the ORF (designated mspA, for Mucoidy-associated Secreted Protein) was expressed in mid- and late-exponential phase in a DeltacsrRS(-)mutant. The presence of an in-frame deletion in mspA did not affect colony appearance, mucoidy or in vitro growth, and there was no difference between DeltamspA and wild-type strains in a mouse model of skin infection. MspA is co-regulated with other factors required for dermonecrosis (e.g. capsule, streptolysin S and purogenic exotoxin B); however, deletion of this gene does not affect expression of hyaluronic acid capsule or severity of skin infection in mice. PMID:11453703

  7. High mobility group protein 1: A collaborator in nucleosome dynamics and estrogen-responsive gene expression.

    PubMed

    Scovell, William M

    2016-05-26

    High mobility group protein 1 (HMGB1) is a multifunctional protein that interacts with DNA and chromatin to influence the regulation of transcription, DNA replication and repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, adenosine triphosphate-independent manner. As a result, the canonical nucleosome is converted to two stable, physically distinct nucleosome conformers. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N' and N'') remain stable and exhibit a number of characteristics that are distinctly different from the canonical nucleosome. These findings complement previous studies that showed (1) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (2) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that a major facet of the mechanism of HMGB1 action involves a restructuring of aspects of the nucleosome that appear to relax structural constraints within the nucleosome. The findings are extended to reveal the differences between ER and the other steroid hormone receptors. A working proposal outlines mechanisms that highlight the multiple facets that HMGB1 may utilize in restructuring the nucleosome. PMID:27247709

  8. Linkage Groups of Protein-Coding Genes in Western Palearctic Water Frogs Reveal Extensive Evolutionary Conservation

    PubMed Central

    Hotz, H.; Uzzell, T.; Berger, L.

    1997-01-01

    Among progeny of a hybrid (Rana shqiperica X R. lessonae) X R. lessonae, 14 of 22 loci form four linkage groups (LGs): (1) mitochondrial aspartate aminotransferase, carbonate dehydratase-2, esterase 4, peptidase D; (2) mannosephosphate isomerase, lactate dehydrogenase-B, sex, hexokinase-1, peptidase B; (3) albumin, fructose-biphosphatase-1, guanine deaminase; (4) mitochondrial superoxide dismutase, cytosolic malic enzyme, xanthine oxidase. Fructose-biphosphate aldolase-2 and cytosolic aspartate aminotransferase possibly form a fifth LG. Mitochondrial aconitate hydratase, α-glucosidase, glyceraldehyde-3-phosphate dehydrogenase, phosphogluconate dehydrogenase, and phosphoglucomutase-2 are unlinked to other loci. All testable linkages (among eight loci of LGs 1, 2, 3, and 4) are shared with eastern Palearctic water frogs. Including published data, 44 protein loci can be assigned to 10 of the 13 chromosomes in Holarctic Rana. Of testable pairs among 18 protein loci, agreement between Palearctic and Nearctic Rana is complete (125 unlinked, 14 linked pairs among 14 loci of five syntenies), and Holarctic Rana and Xenopus laevis are highly concordant (125 shared nonlinkages, 13 shared linkages, three differences). Several Rana syntenies occur in mammals and fish. Many syntenies apparently have persisted for 60-140 X 10(6) years (frogs), some even for 350-400 X 10(6) years (mammals and teleosts). PMID:9286685

  9. Identification of a specific protein factor defective in group A xeroderma pigmentosum cells.

    PubMed

    Sugano, T; Uchida, T; Yamaizumi, M

    1991-10-01

    A protein factor which corrects the defect in xeroderma pigmentosum cells belonging to complementation group A (XP-A cells) was detected in a cell extract prepared from calf thymus. The activity of this factor was measured as the amount of unscheduled DNA synthesis (UDS) reappearing in UV-irradiated XP-A cells after microinjection of the extract. The native molecular mass of this factor was estimated to be 80 kDa by gel-filtration and 25 kDa by glycerol gradient centrifugation. The activity was, however, recovered at a position corresponding to 43 kDa after renaturation on an SDS-PAGE gel. The isoelectric point was determined to be approximately 7.5 by measuring the activity after renaturation on an IEF gel. These values were obtained with a partially purified sample. A spot corresponding to these values was detected on two-dimensional gel electrophoresis with a highly purified sample recovered from an SDS-PAGE gel. The purified protein stimulated UDS specifically in the XP-A cells and endowed the cells with a normal level of UV-resistance. The XP-A cells injected with the factor also showed a normal level of UDS after treatment with either 4HAQO or psoralen plus UV-A. This factor (XP-A complementing factor; XP-ACF) may be involved in the repair of DNA damage induced by various agents. PMID:1778992

  10. Nucleoplasmic Lamin A/C and Polycomb group of proteins: An evolutionarily conserved interplay

    PubMed Central

    Marullo, F.; Cesarini, E.; Antonelli, L.; Gregoretti, F.; Oliva, G.; Lanzuolo, C.

    2016-01-01

    ABSTRACT Nuclear lamins are the main components of the nuclear lamina at the nuclear periphery, providing mechanical support to the nucleus. However, recent findings suggest that lamins also reside in the nuclear interior, as a distinct and dynamic pool with critical roles in transcriptional regulation. In our work we found a functional and evolutionary conserved crosstalk between Lamin A/C and the Polycomb group (PcG) of proteins, this being required for the maintenance of the PcG repressive functions. Indeed, Lamin A/C knock-down causes PcG foci dispersion and defects in PcG-mediated higher order structures, thereby leading to impaired PcG mediated transcriptional repression. By using ad-hoc algorithms for image analysis and PLA approaches we hereby show that PcG proteins are preferentially located in the nuclear interior where they interact with nucleoplasmic Lamin A/C. Taken together, our findings suggest that nuclear components, such as Lamin A/C, functionally interact with epigenetic factors to ensure the correct transcriptional program maintenance. PMID:26930442