TAF(II)170 interacts with the concave surface of TATA-binding protein to inhibit its DNA binding activity.

PubMed

Pereira, L A; van der Knaap, J A; van den Boom, V; van den Heuvel, F A; Timmers, H T

2001-11-01

The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAF(II)170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAF(II)170. We have defined the TBP interaction domain of TAF(II)170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBP(AS)) containing a triple mutation in the concave surface is defective for binding the TAF(II)170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAF(II)170 residues 290 to 381 can inhibit the interaction between Drosophila TAF(II)230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAF(II)170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBP(AS) mutant is less sensitive to TAF(II)170 inhibition. Collectively, our results support a mechanism in which TAF(II)170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface.

Lead inhibition of DNA-binding mechanism of Cys(2)His(2) zinc finger proteins.

PubMed

Hanas, J S; Rodgers, J S; Bantle, J A; Cheng, Y G

1999-11-01

The association of lead with chromatin in cells suggests that deleterious metal effects may in part be mediated through alterations in gene function. To elucidate if and how lead may alter DNA binding of cysteine-rich zinc finger proteins, lead ions were analyzed for their ability to alter the DNA binding mechanism of the Cys(2)His(2) zinc finger protein transcription factor IIIA (TFIIIA). As assayed by DNase I protection, the interaction of TFIIIA with the 50-bp internal control region of the 5S ribosomal gene was partially inhibited by 5 microM lead ions and completely inhibited by 10 to 20 microM lead ions. Preincubation of free TFIIIA with lead resulted in DNA-binding inhibition, whereas preincubation of a TFIIIA/5S RNA complex with lead did not result in DNA-binding inhibition. Because 5S RNA binds TFIIIA zinc fingers, this result is consistent with an inhibition mechanism via lead binding to zinc fingers. The complete loss of DNase I protection on the 5S gene indicates the mechanism of inhibition minimally involves the N-terminal fingers of TFIIIA. Inhibition was not readily reversible and occurred in the presence of an excess of beta-mercaptoethanol. Inhibition kinetics were fast, progressing to completion in approximately 5 min. Millimolar concentrations of sulfhydryl-specific arsenic ions were not inhibitory for TFIIIA binding. Micromolar concentrations of lead inhibited DNA binding by Sp1, another Cys(2)His(2) finger protein, but not by the nonfinger protein AP2. Inhibition of Cys(2)His(2) zinc finger transcription factors by lead ions at concentrations near those known to have deleterious physiological effects points to new molecular mechanisms for lead toxicity in promoting disease.

Characterization and autoradiographic localization of neurotensin binding sites in human sigmoid colon.

PubMed

Azriel, Y; Burcher, E

2001-06-01

Radioiodinated neurotensin ((125)I-NT) was used to characterize and localize NT binding sites in normal human sigmoid colon. Specimens were obtained from patients (30-77 years old) undergoing resection for colon carcinoma. Specific binding of (125)I-NT to sigmoid circular muscle membranes was enhanced by o-phenanthroline (1 mM) but other peptidase inhibitors were ineffective. (125)I-NT bound to a high-affinity site of K(d) = 0.88 +/- 0.09 nM and B(max) = 4.03 +/- 0.66 fmol/mg of wet weight tissue (n = 14), although in the majority of patients another site, of low but variable affinity, could also be detected. Specific binding of 50 pM (125)I-NT was inhibited by NT(8-13) > NT > SR142948A > or = neuromedin N > or = SR48692, consistent with binding to the NT1 receptor. In autoradiographic studies, dense specific binding of (125)I-NT was seen over myenteric and submucosal ganglia, moderate binding over circular muscle, and sparse binding over longitudinal muscle and taenia coli. Levocabastine, which has affinity for the NT2 receptor, did not inhibit specific binding of (125)I-NT in membrane competition or autoradiographic studies. NT contracted sigmoid colon circular muscle strips with a pD(2) value of 6.8 +/- 0.2 nM (n = 25). The contractile responses to NT were significantly potentiated in the presence of tetrodotoxin (1 microM), indicating a neural component. Results from functional studies support actions for NT on both muscle and enteric neurons, consistent with the presence of NT receptors on circular muscle and ganglia of human sigmoid colon. The lack of inhibition by levocabastine suggests that the second binding site detected does not correspond to the NT2 receptor.

Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

PubMed

Ho, Hsu-Tso; Fan, Li; Nowicka-Sans, Beata; McAuliffe, Brian; Li, Chang-Ben; Yamanaka, Gregory; Zhou, Nannan; Fang, Hua; Dicker, Ira; Dalterio, Richard; Gong, Yi-Fei; Wang, Tao; Yin, Zhiwei; Ueda, Yasutsugu; Matiskella, John; Kadow, John; Clapham, Paul; Robinson, James; Colonno, Richard; Lin, Pin-Fang

2006-04-01

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bidlack, J.M.; Frey, D.K.; Seyed-Mozaffari, A.

The binding properties of 14{beta}-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM ({sup 3}H)(D-Ala{sup 2},(Me)Phe{sup 4},Gly(ol){supmore » 5})enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the {mu} binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The {mu} receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the {delta}-selective peptide ({sup 3}H)(D-penicillamine{sup 2},D-penicillamine{sup 5})enkephalin (DPDPE) and (-)-({sup 3}H)bremazocine in the presence of {mu} and {delta} blockers, selective for {kappa} binding sites. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the {mu}s site did not afford protection. These studies have demonstrated that when a disulfide bond at or near {mu} opioid binding sites was reduced, BAM could then alkylate this site, resulting in the specific irreversible labeling of {mu} opioid receptors.« less

Conserved and Variant Epitopes of Plasmodium vivax Duffy Binding Protein as Targets of Inhibitory Monoclonal Antibodies

PubMed Central

Ntumngia, Francis B.; Schloegel, Jesse; Barnes, Samantha J.; McHenry, Amy M.; Singh, Sanjay; King, Christopher L.

2012-01-01

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains. PMID:22215740

Conserved and variant epitopes of Plasmodium vivax Duffy binding protein as targets of inhibitory monoclonal antibodies.

PubMed

Ntumngia, Francis B; Schloegel, Jesse; Barnes, Samantha J; McHenry, Amy M; Singh, Sanjay; King, Christopher L; Adams, John H

2012-03-01

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains.

Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: Inactivation of specific transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fradkin, L.G.; Yoshinaga, S.K.; Berk, A.J.

1987-11-01

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription ofmore » RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoted, however, was not altered by infection of cells with the virus. The authors conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirtus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.« less

Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding.

PubMed

Ailte, Ieva; Lingelem, Anne Berit Dyve; Kavaliauskiene, Simona; Bergan, Jonas; Kvalvaag, Audun Sverre; Myrann, Anne-Grethe; Skotland, Tore; Sandvig, Kirsten

2016-07-26

Shiga toxin (Stx), an AB5 toxin, binds specifically to the neutral glycosphingolipid Gb3 at the cell surface before being transported into cells. We here demonstrate that addition of conical lysophospholipids (LPLs) with large head groups inhibit Stx binding to cells whereas LPLs with small head groups do not. Lysophosphatidylinositol (LPI 18:0), the most efficient LPL with the largest head group, was selected for in-depth investigations to study how the binding of Stx is regulated. We show that the inhibition of Stx binding by LPI is reversible and possibly regulated by cholesterol since addition of methyl-β-cyclodextrin (mβCD) reversed the ability of LPI to inhibit binding. LPI-induced inhibition of Stx binding is independent of signalling and membrane turnover as it occurs in fixed cells as well as after depletion of cellular ATP. Furthermore, data obtained with fluorescent membrane dyes suggest that LPI treatment has a direct effect on plasma membrane lipid packing with shift towards a liquid disordered phase in the outer leaflet, while lysophosphoethanolamine (LPE), which has a small head group, does not. In conclusion, our data show that cellular treatment with conical LPLs with large head groups changes intrinsic properties of the plasma membrane and modulates Stx binding to Gb3.

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site.

PubMed

Ojelabi, Ogooluwa A; Lloyd, Kenneth P; Simon, Andrew H; De Zutter, Julie K; Carruthers, Anthony

2016-12-23

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with K i (app) = 6 μm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with K i (app) = 0.3 μm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site*

PubMed Central

Ojelabi, Ogooluwa A.; Lloyd, Kenneth P.; Simon, Andrew H.; De Zutter, Julie K.; Carruthers, Anthony

2016-01-01

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 μm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 μm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites. PMID:27836974

Replication of damaged DNA in vitro is blocked by p53

PubMed Central

Zhou, Jianmin; Prives, Carol

2003-01-01

The tumor suppressor protein p53 may have other roles and functions in addition to its well-documented ability to serve as a sequence-specific transcriptional activator in response to DNA damage. We showed previously that p53 can block the replication of polyomavirus origin-containing DNA (Py ori-DNA) in vitro when p53 binding sites are present on the late side of the Py ori. Here we have both further extended these observations and have also examined whether p53 might be able to bind directly to and inhibit the replication of damaged DNA. We found that p53 strongly inhibits replication of γ-irradiated Py ori-DNA and such inhibition requires both the central DNA binding domain and the extreme C-terminus of the p53 protein. An endogenous p53 binding site lies within the Py origin and is required for the ability of p53 to block initiation of replication from γ-irradiated Py ori-DNA, suggesting the possibility of DNA looping caused by p53 binding both non-specifically to sites of DNA damage and specifically to the endogenous site in the polyomavirus origin. Our results thus suggest the possibility that under some circumstances p53 might serve as a direct regulator of DNA replication and suggest as well an additional function for cooperation between its two autonomous DNA binding domains. PMID:12853603

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

PubMed

Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

2009-08-15

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

Oxidative phosphorylation. The relation between the specific binding of trimethlytin and triethyltin to mitochondria and their effects on various mitochondrial functions

PubMed Central

Aldridge, W. N.; Street, B. W.

1971-01-01

1. A binding site (site 1) is present in mitochondria with affinity for trimethyltin and triethyltin adequate for a site to which they could be attached when the processes of energy conservation are inhibited. 2. The quantitative relationships between the binding of trimethyltin and triethyltin to site 1 and their effects on various mitochondrial functions have been examined. 3. ATP synthesis linked to the oxidation of pyruvate, succinate and intramitochondrial substrate, ATP synthesis and oxygen uptake (succinate or pyruvate as substrate) stimulated by uncoupling agents are all inhibited by trimethyltin and triethyltin; when inhibition is less than 50% the ratio (percentage inhibition)/(percentage of binding site 1 complexed) is approx. 10:1. 4. ATP synthesis linked to the oxidation of reduced cytochrome c (ascorbate+NNN′N′-tetramethyl-p-phenylenediamine), ATP hydrolysis and oxygen uptake in the presence of low concentrations of trimethyltin and triethyltin approach zero activity as the proportion of binding site 1 complexed approaches 100%. 5. Possible interpretations of these findings are discussed with reference to published arrangements for coupling of electron transport to ATP synthesis and also to our present knowledge of the chemical and biological specificity of trialkyltin compounds. PMID:5126473

Nuclear TRIM25 Specifically Targets Influenza Virus Ribonucleoproteins to Block the Onset of RNA Chain Elongation.

PubMed

Meyerson, Nicholas R; Zhou, Ligang; Guo, Yusong R; Zhao, Chen; Tao, Yizhi J; Krug, Robert M; Sawyer, Sara L

2017-11-08

TRIM25 is an E3 ubiquitin ligase that activates RIG-I to promote the antiviral interferon response. The NS1 protein from all strains of influenza A virus binds TRIM25, although not all virus strains block the interferon response, suggesting alternative mechanisms for TRIM25 action. Here we present a nuclear role for TRIM25 in specifically restricting influenza A virus replication. TRIM25 inhibits viral RNA synthesis through a direct mechanism that is independent of its ubiquitin ligase activity and the interferon pathway. This activity can be inhibited by the viral NS1 protein. TRIM25 inhibition of viral RNA synthesis results from its binding to viral ribonucleoproteins (vRNPs), the structures containing individual viral RNA segments, the viral polymerase, and multiple viral nucleoproteins. TRIM25 binding does not inhibit initiation of capped-RNA-primed viral mRNA synthesis by the viral polymerase. Rather, the onset of RNA chain elongation is inhibited because TRIM25 prohibits the movement of RNA into the polymerase complex. Copyright © 2017 Elsevier Inc. All rights reserved.

Intragenic DNA methylation and BORIS-mediated cancer-specific splicing contribute to the Warburg effect

PubMed Central

Singh, Smriti; Narayanan, Sathiya Pandi; Biswas, Kajal; Gupta, Amit; Ahuja, Neha; Yadav, Sandhya; Panday, Rajendra Kumar; Samaiya, Atul; Sharan, Shyam K.

2017-01-01

Aberrant alternative splicing and epigenetic changes are both associated with various cancers, but epigenetic regulation of alternative splicing in cancer is largely unknown. Here we report that the intragenic DNA methylation-mediated binding of Brother of Regulator of Imprinted Sites (BORIS) at the alternative exon of Pyruvate Kinase (PKM) is associated with cancer-specific splicing that promotes the Warburg effect and breast cancer progression. Interestingly, the inhibition of DNA methylation, BORIS depletion, or CRISPR/Cas9-mediated deletion of the BORIS binding site leads to a splicing switch from cancer-specific PKM2 to normal PKM1 isoform. This results in the reversal of the Warburg effect and the inhibition of breast cancer cell growth, which may serve as a useful approach to inhibit the growth of breast cancer cells. Importantly, our results show that in addition to PKM splicing, BORIS also regulates the alternative splicing of several genes in a DNA methylation-dependent manner. Our findings highlight the role of intragenic DNA methylation and DNA binding protein BORIS in cancer-specific splicing and its role in tumorigenesis. PMID:29073069

Complex high affinity interactions occur between MHCI and superantigens

NASA Technical Reports Server (NTRS)

Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

1998-01-01

Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

Multi-specificity of a Psathyrella velutina mushroom lectin: heparin/pectin binding occurs at a site different from the N-acetylglucosamine/N-acetylneuraminic acid-specific site.

PubMed

Ueda, H; Saitoh, T; Kojima, K; Ogawa, H

1999-09-01

An N-acetylglucosamine (GlcNAc)/N-acetylneuraminic acid-specific lectin from the fruiting body of Psathyrella velutina (PVL) is a useful probe for the detection and fractionation of specific carbohydrates. In this study, PVL was found to exhibit multispecificity to acidic polysaccharides and sulfatides. Purified PVL and a counterpart lectin to PVL in the mycelium interact with heparin neoproteoglycans, as detected by both membrane analysis and solid phase assay. The pH-dependencies of the binding to heparin and GlcNAc5-6 differ. The heparin binding of PVL is inhibited best by pectin, polygalacturonic acid, and highly sulfated polysaccharides, but not by GlcNAc, colominic acid, or other glycosaminoglycans. Sandwich affinity chromatography indicated that PVL can simultaneously interact with heparin- and GlcNAc-containing macromolecules. Extensive biotinylation was found to suppress the binding activity to heparin while the GlcNAc binding activity is retained. On the other hand, biotinyl PVL binds to sulfatide and the binding is not inhibited by GlcNAc, N-acetylneuraminic acid, or heparin. These results indicate that PVL is a multi-ligand adhesive lectin that can interact with various glycoconjugates. This multispecificity needs to be recognized when using PVL as a sugar-specific probe to avoid misleading information about the nature of glycoforms.

Viral and Cellular Determinants of the Hepatitis C Virus Envelope-Heparan Sulfate Interaction▿

PubMed Central

Barth, Heidi; Schnober, Eva K.; Zhang, Fuming; Linhardt, Robert J.; Depla, Erik; Boson, Bertrand; Cosset, Francois-Loic; Patel, Arvind H.; Blum, Hubert E.; Baumert, Thomas F.

2006-01-01

Cellular binding and entry of hepatitis C virus (HCV) are the first steps of viral infection and represent a major target for antiviral antibodies and novel therapeutic strategies. We have recently demonstrated that heparan sulfate (HS) plays a key role in the binding of HCV envelope glycoprotein E2 to target cells (Barth et al., J. Biol. Chem. 278:41003-41012, 2003). In this study, we characterized the HCV-HS interaction and analyzed its inhibition by antiviral host immune responses. Using recombinant envelope glycoproteins, virus-like particles, and HCV pseudoparticles as model systems for the early steps of viral infection, we mapped viral and cellular determinants of HCV-HS interaction. HCV-HS binding required a specific HS structure that included N-sulfo groups and a minimum of 10 to 14 saccharide subunits. HCV envelope binding to HS was mediated by four viral epitopes overlapping the E2 hypervariable region 1 and E2-CD81 binding domains. In functional studies using HCV pseudoparticles, we demonstrate that HCV binding and entry are specifically inhibited by highly sulfated HS. Finally, HCV-HS binding was markedly inhibited by antiviral antibodies derived from HCV-infected individuals. In conclusion, our results demonstrate that binding of the viral envelope to a specific HS configuration represents an important step for the initiation of viral infection and is a target of antiviral host immune responses in vivo. Mapping of viral and cellular determinants of HCV-HS interaction sets the stage for the development of novel HS-based antiviral strategies targeting viral attachment and entry. PMID:16928753

IgE binding to peanut allergens is inhibited by combined D-aspartic and D-glutamic acids.

PubMed

Chung, Si-Yin; Reed, Shawndrika

2015-01-01

The objective of this study was to determine if D-amino acids (D-aas) bind and inhibit immunoglobulin E (IgE) binding to peanut allergens. D-aas such as D-Asp (aspartic acid), D-Glu (glutamic acid), combined D-[Asp/Glu] and others were each prepared in a cocktail of 9 other D-aas, along with L-amino acids (L-aas) and controls. Each sample was mixed with a pooled plasma from peanut-allergic donors, and tested by ELISA (enzyme-linked immunosorbent assay) and Western blots for IgE binding to peanut allergens. Results showed that D-[Asp/Glu] (4 mg/ml) inhibited IgE binding (75%) while D-Glu, D-Asp and other D-aas had no inhibitory effect. A higher inhibition was seen with D-[Asp/Glu] than with L-[Asp/Glu]. We concluded that IgE was specific for D-[Asp/Glu], not D-Asp or D-Glu, and that D-[Asp/Glu] was more reactive than was L-[Asp/Glu] in IgE inhibition. The finding indicates that D-[Asp/Glu] may have the potential for removing IgE or reducing IgE binding to peanut allergens in vitro. Published by Elsevier Ltd.

Modification of Herbicide Binding to Photosystem II in Two Biotypes of Senecio vulgaris L

PubMed Central

Pfister, Klaus; Radosevich, Steven R.; Arntzen, Charles J.

1979-01-01

The present study compares the binding and inhibitory activity of two photosystem II inhibitors: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron [DCMU]) and 2-chloro-4-(ethylamine)-6-(isopropyl amine)-S-triazene (atrazine). Chloroplasts isolated from naturally occurring triazine-susceptible and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.) showed the following characteristics. (a) Diuron strongly inhibited photosynthetic electron transport from H2O to 2,6-dichlorophenolindophenol in both biotypes. Strong inhibition by atrazine was observed only with the susceptible chloroplasts. (b) Hill plots of electron transport inhibition data indicate a noncooperative binding of one inhibitor molecule at the site of action for both diuron and atrazine. (c) Susceptible chloroplasts show a strong diuron and atrazine binding (14C-radiolabel assays) with binding constants (K) of 1.4 × 10−8 molar and 4 × 10−8 molar, respectively. In the resistant chloroplasts the diuron binding was slightly decreased (K = 5 × 10−8 molar), whereas no specific atrazine binding was detected. (d) In susceptible chloroplasts, competitive binding between radioactively labeled diuron and non-labeled atrazine was observed. This competition was absent in the resistant chloroplasts. We conclude that triazine resistance of both intact plants and isolated chloroplasts of Senecio vulgaris L. is based upon a minor modification of the protein in the photosystem II complex which is responsible for herbicide binding. This change results in a specific loss of atrazine (triazine)-binding capacity. PMID:16661120

Characterization of the interaction of yeast enolase with polynucleotides.

PubMed

al-Giery, A G; Brewer, J M

1992-09-23

Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.

BLM and RMI1 alleviate RPA inhibition of TopoIIIα decatenase activity.

PubMed

Yang, Jay; Bachrati, Csanad Z; Hickson, Ian D; Brown, Grant W

2012-01-01

RPA is a single-stranded DNA binding protein that physically associates with the BLM complex. RPA stimulates BLM helicase activity as well as the double Holliday junction dissolution activity of the BLM-topoisomerase IIIα complex. We investigated the effect of RPA on the ssDNA decatenase activity of topoisomerase IIIα. We found that RPA and other ssDNA binding proteins inhibit decatenation by topoisomerase IIIα. Complex formation between BLM, TopoIIIα, and RMI1 ablates inhibition of decatenation by ssDNA binding proteins. Together, these data indicate that inhibition by RPA does not involve species-specific interactions between RPA and BLM-TopoIIIα-RMI1, which contrasts with RPA modulation of double Holliday junction dissolution. We propose that topoisomerase IIIα and RPA compete to bind to single-stranded regions of catenanes. Interactions with BLM and RMI1 enhance toposiomerase IIIα activity, promoting decatenation in the presence of RPA.

Molecular mechanism of transcription inhibition by phage T7 gp2 protein.

PubMed

Mekler, Vladimir; Minakhin, Leonid; Sheppard, Carol; Wigneshweraraj, Sivaramesh; Severinov, Konstantin

2011-11-11

Escherichia coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). gp2 inhibits formation of open promoter complex by binding to the β' jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay, we performed quantitative real-time measurements of specific binding of the RNAP-gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β' jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the -10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β' jaw. Copyright © 2011 Elsevier Ltd. All rights reserved.

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Specific high-affinity binding sites for a synthetic gliadin heptapeptide of human peripheral blood lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Payan, D.G.; Horvath, K.; Graf, L.

1987-03-23

The synthetic peptide containing residues 43-49 of ..cap alpha..-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. The authors demonstrate using radiolabeled ..cap alpha..-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (K/sub D/) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of ..cap alpha..-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of (/sup 125/I)..cap alpha..-gliadin(43-49) than T-lymphocytes. The lymphocytemore » ..cap alpha..-gliadin(43-49) receptor may play an important role in mediating the immunological response to ..cap alpha..-gliadin. 16 references, 4 figures.« less

Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins

PubMed Central

Chen, Yu; Yang, Fan; Zubovic, Lorena; Pavelitz, Tom; Yang, Wen; Godin, Katherine; Walker, Matthew; Zheng, Suxin; Macchi, Paolo; Varani, Gabriele

2016-01-01

The RNA Recognition Motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with new specificity would provide valuable tools and an exacting test of our understanding of specificity. We have achieved the first successful re-design of the specificity of an RRM using rational methods and demonstrated re-targeting of activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of miR-21 precursor with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications. PMID:27428511

Inhibition of /sup 3/H-leukotriene D4 binding to guinea pig lung receptors by the novel leukotriene antagonist ICI 198,615

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aharony, D.; Falcone, R.C.; Krell, R.D.

1987-12-01

The specific binding of (/sup 3/H)5(S)hydroxy-6(R)-S-cysteinylglycyl -7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid ((/sup 3/H)LTD4) to receptors on guinea pig lung parenchymal membranes and its inhibition by ICI 198,615, a representative example of a new class of leukotriene antagonists, was characterized by a receptor-ligand binding assay. (/sup 3/H)LTD4 bound specifically and rapidly (Kon = 0.29 +/- 0.6 nM-1.min-1) reaching equilibrium within 15 min. The rate of binding was greatly inhibited in the presence of ICI 198,615. Excess LTD4 or ICI 198,615 slowly (t1/2 = 20 min) dissociated about 70% of the receptor-bound (/sup 3/H)LTD4, whereas in combination with GTP analogs, both induced a rapid (t1/2more » less than 5 min) and full dissociation. Equilibrium saturation analysis of (/sup 3/H)LTD4 binding demonstrated a saturable (Bmax = 1014 +/- 174 fmol/mg) and high affinity (Kd = 0.43 +/- 0.09 nM) binding site. A high degree of stereoselectivity was demonstrated with inhibition of binding by the stereoisomers of LTD4: S,R much greater than R,R greater than R,S much greater than S,S. The rank order for inhibition of binding by peptide leukotriene was: LTD4 greater than 5(S)-hydroxy-6(R)-S-cysteinyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid much greater than 5(S)hydroxy-6(R)-S-glutathionyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (potency ratios were: 1:4:590). In competition assays, ICI 198,615 competitively inhibited binding of (/sup 3/H)LTD4 (Ki = 0.27 +/- 0.16 nM) and was 2300-fold and 3100-fold more potent than LY171883 or FPL55712. These data, together with results obtained previously in functional receptor assays, illustrate that this new class of leukotriene antagonists are the most potent and selective competitive antagonists of LTD4 receptors yet described.« less

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver.

PubMed

Yachi, K; Sugiyama, Y; Sawada, Y; Iga, T; Ikeda, Y; Toda, G; Hanano, M

1989-01-16

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants (Kd = 0.1-0.12 microM) and the binding capacities (n = 11-15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5-2.5 and 100 microM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

The correlation between ouabain binding and potassium pump inhibition in human and sheep erythrocytes.

PubMed Central

Joiner, C H; Lauf, P K

1978-01-01

1. [3H]Ouabain binding to human and sheep red blood cells was shown to be specific for receptors associated with Na/K transport. Virtually all tritium binding was abolished by dilution with unlabelled drug. Saturation levels of binding were independent of glycoside concentration and were identical to those associated with 100% inhibition of K pumping. 2. [3H]Ouabain binding and 42K influx were measured simultaneously in order to correlate the degree of K pump inhibition with the amount of glycoside bound. Results by this method agreed exactly with those obtained by pre-exposing cells to drug, followed by washing and then measuring K influx. 3. Plots of [3H]oubain binding vs. K pump inhibition were rectilinear for human and low K (LK) sheep red cells, indicating one glycoside receptor per K pump site and functional homogeneity of pump sites. High K (HK) sheep red cells exhibited curved plots of binding versus inhibition, which were best explained in terms of one receptor per pump, but a heterogeneous population of pump sites. 4. External K reduced the rate of glycoside binding, but did not alter the relationship between binding and inhibition. 5. The number of K pump sites was estimated as 450--500 per human cell and 30--50 per LK sheep cell. HK sheep cells had 90--130 sites per cell, of which eighty to ninety were functionally dominant. The number of K pump sites on LK sheep cells was not changed by anti-L, although the maximum velocity of pump turnover was increased. PMID:722573

Identification of a receptor for ADP on blood platelets by photoaffinity labelling.

PubMed Central

Cristalli, G; Mills, D C

1993-01-01

The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5'-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase. Images Figure 5 PMID:8387782

Functional analysis of CedA based on its structure: residues important in binding of DNA and RNA polymerase and in the cell division regulation

PubMed Central

Abe, Yoshito; Fujisaki, Naoki; Miyoshi, Takanori; Watanabe, Noriko; Katayama, Tsutomu; Ueda, Tadashi

2016-01-01

DnaAcos, a mutant of the initiator DnaA, causes overinitiation of chromosome replication in Escherichia coli, resulting in inhibition of cell division. CedA was found to be a multi-copy suppressor which represses the dnaAcos inhibition of cell division. However, functional mechanism of CedA remains elusive except for previously indicated possibilities in binding to DNA and RNA polymerase. In this study, we searched for the specific sites of CedA in binding of DNA and RNA polymerase and in repression of cell division inhibition. First, DNA sequence to which CedA preferentially binds was determined. Next, the several residues and β4 region in CedA C-terminal domain was suggested to specifically interact with the DNA. Moreover, we found that the flexible N-terminal region was required for tight binding to longer DNA as well as interaction with RNA polymerase. Based on these results, several cedA mutants were examined in ability for repressing dnaAcos cell division inhibition. We found that the N-terminal region was dispensable and that Glu32 in the C-terminal domain was required for the repression. These results suggest that CedA has multiple roles and residues with different functions are positioned in the two regions. PMID:26400504

Isolation and characterization of iron chelators from turmeric (Curcuma longa): selective metal binding by curcuminoids.

PubMed

Messner, Donald J; Surrago, Christine; Fiordalisi, Celia; Chung, Wing Yin; Kowdley, Kris V

2017-10-01

Iron overload disorders may be treated by chelation therapy. This study describes a novel method for isolating iron chelators from complex mixtures including plant extracts. We demonstrate the one-step isolation of curcuminoids from turmeric, the medicinal food spice derived from Curcuma longa. The method uses iron-nitrilotriacetic acid (NTA)-agarose, to which curcumin binds rapidly, specifically, and reversibly. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin each bound iron-NTA-agarose with comparable affinities and a stoichiometry near 1. Analyses of binding efficiencies and purity demonstrated that curcuminoids comprise the primary iron binding compounds recovered from a crude turmeric extract. Competition of curcuminoid binding to the iron resin was used to characterize the metal binding site on curcumin and to detect iron binding by added chelators. Curcumin-Iron-NTA-agarose binding was inhibited by other metals with relative potency: (>90% inhibition) Cu 2+  ~ Al 3+  > Zn 2+  ≥ Ca 2+  ~ Mg 2+  ~ Mn 2+ (<20% inhibition). Binding was also inhibited by pharmaceutical iron chelators (desferoxamine or EDTA) or by higher concentrations of weak iron chelators (citrate or silibinin). Investigation of the physiological effects of iron binding by curcumin revealed that curcumin uptake by cultured cells was reduced >80% by addition of iron to the media; uptake was completely restored by desferoxamine. Ranking of metals by relative potencies for blocking curcumin uptake agreed with their relative potencies in blocking curcumin binding to iron-NTA-agarose. We conclude that curcumin can selectively bind toxic metals including iron in a physiological setting, and propose inhibition of curcumin binding to iron-NTA-agarose for iron chelator screening.

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

PubMed Central

1984-01-01

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron- glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells. PMID:6725397

Precipitation of the thyrotropin receptor and identification of thyroid autoantigens using Graves' disease immunoglobulins.

PubMed Central

Heyma, P; Harrison, L C

1984-01-01

The thyrotropin (TSH) receptor is a putative target for autoantibodies in Graves' hyperthyroidism and therefore, should be capable of being identified, isolated, and structurally characterized by immunological means. To this end, four sera from patients with hyperthyroidism, three of which inhibited the binding of 125I-TSH to Triton-solubilized human thyroid membranes, were used to isolate TSH receptors by immunoprecipitation. To account for an effect of TSH binding or receptor occupancy on the ability of Graves' immunoglobulins to precipitate TSH receptors, two approaches were taken: (a) specific 125I-TSH binding activity was measured after solubilized thyroid membranes had been incubated with Graves' sera followed by precipitation with Staphylococcus protein A ("receptor depletion"); (b) TSH binding sites were labeled with 125I-TSH and the complexes were precipitated using Graves' sera and Staphylococcus protein A ("receptor precipitation"). The three sera which inhibited 125I-TSH binding depleted 125I-TSH binding activity between 30-80%. Preformed complexes between Staphylococcus protein A and immunoglobulins in these sera were also able to deplete 125I-TSH binding activity. However, after receptor depletion, the one serum that did not inhibit 125I-TSH binding was associated with a significant increase in 125I-TSH binding. All four sera specifically precipitated 80-100% of receptors identified by prelabeling with 125I-TSH. The dilutions of sera that precipitated 50% of 125I-TSH-receptor complexes ranged from 1:150-1:20. Complexes were partially precipitated by high concentrations of control sera (1:20), but the relative potency of control sera was at least fourfold less than Graves' sera. Immunoprecipitates of 125I-labeled thyroid membranes were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography to reveal Graves'-specific bands of reduced molecular weights of 100-110,000, 80-90,000, and 70-75,000. These bands were similar to those obtained from 125I-labeled thyroid membranes purified by TSH affinity chromatography. Thus, Graves' immunoglobulins: (a) precipitate unoccupied and occupied TSH receptors, (b) in one case, neither inhibit binding nor immunodeplete the unoccupied receptor but immunoprecipitate 125I-TSH-receptor complexes, suggesting that binding of TSH may initiate an interaction between the binding site and a separate immunoreactive molecule, and (c) identify the molecular structure of Graves' autoantigens, putatively, the TSH receptor. Images PMID:6088581

Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles

PubMed Central

1994-01-01

Elevation of cAMP can cause gene-specific inhibition of interleukin 2 (IL-2) expression. To investigate the mechanism of this effect, we have combined electrophoretic mobility shift assays and in vivo genomic footprinting to assess both the availability of putative IL-2 transcription factors in forskolin-treated cells and the functional capacity of these factors to engage their sites in vivo. All observed effects of forskolin depended upon protein kinase A, for they were blocked by introduction of a dominant negative mutant subunit of protein kinase A. In the EL4.E1 cell line, we report specific inhibitory effects of cAMP elevation both on NF-kappa B/Rel family factors binding at -200 bp, and on a novel, biochemically distinct "TGGGC" factor binding at -225 bp with respect to the IL-2 transcriptional start site. Neither NF-AT nor AP-1 binding activities are detectably inhibited in gel mobility shift assays. Elevation of cAMP inhibits NF-kappa B activity with delayed kinetics in association with a delayed inhibition of IL-2 RNA accumulation. Activation of cells in the presence of forskolin prevents the maintenance of stable protein- DNA interactions in vivo, not only at the NF-kappa B and TGGGC sites of the IL-2 enhancer, but also at the NF-AT, AP-1, and other sites. This result, and similar results in cyclosporin A-treated cells, imply that individual IL-2 transcription factors cannot stably bind their target sequences in vivo without coengagement of all other distinct factors at neighboring sites. It is proposed that nonhierarchical, cooperative enhancement of binding is a structural basis of combinatorial transcription factor action at the IL-2 locus. PMID:8113685

A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system.

PubMed

D'Souza, Alicia D; Belotserkovskii, Boris P; Hanawalt, Philip C

2018-02-01

The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e.g. secondary promoter) is present upstream from the primary promoter of the target gene. In this case, transcription inhibition might be achieved by inducing the formation of an RNA-DNA hybrid (R-loop) upon transcription from the secondary promoter. The R-loop could extend into the region of the primary promoter, to interfere with promoter recognition by RNA polymerase and thereby inhibit transcription. As a sequence-specific R-loop-inducing agent, a peptide nucleic acid (PNA) could be designed to facilitate R-loop formation by sequestering the non-template DNA strand. To investigate this mode for transcription inhibition, we have employed a model system in which a PNA binding site is localized between the T3 and T7 phage RNA polymerase promoters, which respectively assume the roles of primary and secondary promoters. In accord with our model, we have demonstrated that with PNA-bound DNA substrates, transcription from the T7 promoter reduces transcription from the T3 promoter by 30-fold, while in the absence of PNA binding there is no significant effect of T7 transcription upon T3 transcription. Copyright © 2018 Elsevier B.V. All rights reserved.

Raffinose, a plant galactoside, inhibits Pseudomonas aeruginosa biofilm formation via binding to LecA and decreasing cellular cyclic diguanylate levels

NASA Astrophysics Data System (ADS)

Kim, Han-Shin; Cha, Eunji; Kim, Yunhye; Jeon, Young Ho; Olson, Betty H.; Byun, Youngjoo; Park, Hee-Deung

2016-05-01

Biofilm formation on biotic or abiotic surfaces has unwanted consequences in medical, clinical, and industrial settings. Treatments with antibiotics or biocides are often ineffective in eradicating biofilms. Promising alternatives to conventional agents are biofilm-inhibiting compounds regulating biofilm development without toxicity to growth. Here, we screened a biofilm inhibitor, raffinose, derived from ginger. Raffinose, a galactotrisaccharide, showed efficient biofilm inhibition of Pseudomonas aeruginosa without impairing its growth. Raffinose also affected various phenotypes such as colony morphology, matrix formation, and swarming motility. Binding of raffinose to a carbohydrate-binding protein called LecA was the cause of biofilm inhibition and altered phenotypes. Furthermore, raffinose reduced the concentration of the second messenger, cyclic diguanylate (c-di-GMP), by increased activity of a c-di-GMP specific phosphodiesterase. The ability of raffinose to inhibit P. aeruginosa biofilm formation and its molecular mechanism opens new possibilities for pharmacological and industrial applications.

Endothelial Galectin-1 Binds to Specific Glycans on Nipah Virus Fusion Protein and Inhibits Maturation, Mobility, and Function to Block Syncytia Formation

PubMed Central

Garner, Omai B.; Aguilar, Hector C.; Fulcher, Jennifer A.; Levroney, Ernest L.; Harrison, Rebecca; Wright, Lacey; Robinson, Lindsey R.; Aspericueta, Vanessa; Panico, Maria; Haslam, Stuart M.; Morris, Howard R.; Dell, Anne

2010-01-01

Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell. PMID:20657665

Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain

NASA Astrophysics Data System (ADS)

Miller, Miles A.; Moss, Marcia L.; Powell, Gary; Petrovich, Robert; Edwards, Lori; Meyer, Aaron S.; Griffith, Linda G.; Lauffenburger, Douglas A.

2015-10-01

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

Increase in serotonin 5-HT sub 1A receptors in prefrontal and temporal cortices of brains from patients with chronic schizophrenia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Takeshi; Nishino, Naoki; Nakai, Hisao

1991-01-01

Binding studies with ({sup 3}H)8-hydroxy-2-(di-n-propylamino)tetralin (({sup 3}H)8-OH-DPAT), a specific serotonin{sub 1A} (5-HT{sub 1A}) receptor agonist, were done on the autopsied brains from control subjects and from patients with chronic schizophrenia. In the controls, representative Scatchard plots for the specific ({sup 3}H)8-OH-DPAT bindings in the prefrontal cortex and hippocampus revealed a single component of high affinity binding site. The ({sup 3}H)8-OH-DPAT bindings to the prefrontal cortex and hippocampus were potently inhibited by serotonin and 5-HT{sub 1A} agonists, while other neurotransmitters, 5-HT{sub 2} and 5-HT{sub 3} related compounds did not inhibit the binding. The bindings were decreased in the presence of 0.1mMmore » GTP and 0.1mM GppNHp but not in the presence of 0.1mM GMP. In the prefrontal and temporal cortices of schizophrenics, there was a significant increase in the specific ({sup 3}H)8-OH-DPAT binding, by 40% and 60%, respectively, with no change in the hippocampus, amygdala, cingulum, motor cortex, parietal or occipital cortex, as compared to findings in the controls.« less

Inhibition of Human Papillomavirus Type 16 Infection Using an RNA Aptamer.

PubMed

Valencia-Reséndiz, Diana Gabriela; Palomino-Vizcaino, Giovanni; Tapia-Vieyra, Juana Virginia; Benítez-Hess, María Luisa; Leija-Montoya, Ana Gabriela; Alvarez-Salas, Luis Marat

2018-04-01

Human papillomavirus type 16 (HPV16) DNA has been found in ∼50% of cervical tumors worldwide. HPV infection starts with the binding of the virus capsid to heparan sulfate (HS) receptors exposed on the surface of epithelial basal layer keratinocytes. Previously, our group isolated a high-affinity RNA aptamer (Sc5c3) specific for HPV16 L1 virus-like particles (VLPs). In this study, we report the inhibition of HPV16 infection by Sc5c3 in a pseudovirus (PsVs) model. 293TT cells were infected by HPV16 PsVs containing the yellow fluorescent protein (YFP) as reporter gene. Incubation of HPV16 PsVs with Sc5c3 before infection resulted in a dose-dependent decrease in YFP fluorescence, suggesting infection inhibition. Aptamer degradation by RNase A restored PsVs infectivity, supporting the previous observation that Sc5c3 aptamer can inhibit infection. VLP mutants with removed HS binding sites were used in binding assays to elucidate the Sc5c3 blocking mechanism; however, no binding difference was observed between wild-type and mutant VLPs, suggesting that pseudoinfection inhibition relies on mechanisms additional to electrostatic HS binding site interaction. A DNA/RNA Sc5c3 version also inhibited HPV PsVs infection, suggesting that a modified, nuclease-resistant Sc5c3 may be used to inhibit HPV16 infection in vivo.

The effect of extracts of the roots of the stinging nettle (Urtica dioica) on the interaction of SHBG with its receptor on human prostatic membranes.

PubMed

Hryb, D J; Khan, M S; Romas, N A; Rosner, W

1995-02-01

Extracts from the roots of the stinging nettle (Urtica dioica) are used in the treatment of benign prostatic hyperplasia. The mechanisms underlying this treatment have not been elucidated. We set out to determine whether specific extracts from U. dioica had the ability to modulate the binding of sex hormone-binding globulin to its receptor on human prostatic membranes. Four substances contained in U. dioica were examined: an aqueous extract; an alcoholic extract; U. dioica agglutinin, and stigmasta-4-en-3-one. Of these, only the aqueous extract was active. It inhibited the binding of 125I-SHBG to its receptor. The inhibition was dose related, starting at about 0.6 mg/ml and completely inhibited binding at 10 mg/ml.

Structural basis for subtype-specific inhibition of the P2X7 receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karasawa, Akira; Kawate, Toshimitsu

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of themore » drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.« less

Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth.

PubMed

Vippagunta, S R; Dorn, A; Matile, H; Bhattacharjee, A K; Karle, J M; Ellis, W Y; Ridley, R G; Vennerstrom, J L

1999-11-04

Considerable data now support the hypothesis that chloroquine (CQ)-hematin binding in the parasite food vacuole leads to inhibition of hematin polymerization and parasite death by hematin poisoning. To better understand the structural specificity of CQ-hematin binding, 13 CQ analogues were chosen and their hematin binding affinity, inhibition of hematin polymerization, and inhibition of parasite growth were measured. As determined by isothermal titration calorimetry (ITC), the stoichiometry data and exothermic binding enthalpies indicated that, like CQ, these analogues bind to two or more hematin mu-oxo dimers in a cofacial pi-pi sandwich-type complex. Association constants (K(a)'s) ranged from 0.46 to 2.9 x 10(5) M(-1) compared to 4.0 x 10(5) M(-1) for CQ. Remarkably, we were not able to measure any significant interaction between hematin mu-oxo dimer and 11, the 6-chloro analogue of CQ. This result indicates that the 7-chloro substituent in CQ is a critical structural determinant in its binding affinity to hematin mu-oxo dimer. Molecular modeling experiments reinforce the view that the enthalpically favorable pi-pi interaction observed in the CQ-hematin mu-oxo dimer complex derives from a favorable alignment of the out-of-plane pi-electron density in CQ and hematin mu-oxo dimer at the points of intermolecular contact. For 4-aminoquinolines related to CQ, our data suggest that electron-withdrawing functional groups at the 7-position of the quinoline ring are required for activity against both hematin polymerization and parasite growth and that chlorine substitution at position 7 is optimal. Our results also confirm that the CQ diaminoalkyl side chain, especially the aliphatic tertiary nitrogen atom, is an important structural determinant in CQ drug resistance. For CQ analogues 1-13, the lack of correlation between K(a) and hematin polymerization IC(50) values suggests that other properties of the CQ-hematin mu-oxo dimer complex, rather than its association constant alone, play a role in the inhibition of hematin polymerization. However, there was a modest correlation between inhibition of hematin polymerization and inhibition of parasite growth when hematin polymerization IC(50) values were normalized for hematin mu-oxo dimer binding affinities, adding further evidence that antimalarial 4-aminoquinolines act by this mechanism.

[Effect of calcium and magnesium ions on the interaction of corticosterone with the cytosol receptor(s) in the rat brain].

PubMed

Ueda, M

1981-01-01

The effects of calcium and magnesium ions on the corticosterone binding to rat brain cytosol receptor protein(s) were investigated. The increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, the specific [3H] corticosterone binding increased 1.3-fold and 1.5 respectively. The addition of MnCl2 and KCl did not affect this binding. The binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EDTA and complete inhibition was observed at concentration equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.

Structural analysis of ibuprofen binding to human adipocyte fatty-acid binding protein (FABP4).

PubMed

González, Javier M; Fisher, S Zoë

2015-02-01

Inhibition of human adipocyte fatty-acid binding protein (FABP4) has been proposed as a treatment for type 2 diabetes, fatty liver disease and atherosclerosis. However, FABP4 displays a naturally low selectivity towards hydrophobic ligands, leading to the possibility of side effects arising from cross-inhibition of other FABP isoforms. In a search for structural determinants of ligand-binding selectivity, the binding of FABP4 towards a group of small molecules structurally related to the nonsteroidal anti-inflammatory drug ibuprofen was analyzed through X-ray crystallography. Several specific hydrophobic interactions are shown to enhance the binding affinities of these compounds, whereas an aromatic edge-to-face interaction is proposed to determine the conformation of bound ligands, highlighting the importance of aromatic interactions in hydrophobic environments.

DNA Mismatch Binding and Antiproliferative Activity of Rhodium Metalloinsertors

PubMed Central

Ernst, Russell J.; Song, Hang; Barton, Jacqueline K.

2009-01-01

Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M−1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. PMID:19175313

Parallel Distractor Rejection as a Binding Mechanism in Search

PubMed Central

Dent, Kevin; Allen, Harriet A.; Braithwaite, Jason J.; Humphreys, Glyn W.

2012-01-01

The relatively common experimental visual search task of finding a red X amongst red O’s and green X’s (conjunction search) presents the visual system with a binding problem. Illusory conjunctions (ICs) of features across objects must be avoided and only features present in the same object bound together. Correct binding into unique objects by the visual system may be promoted, and ICs minimized, by inhibiting the locations of distractors possessing non-target features (e.g., Treisman and Sato, 1990). Such parallel rejection of interfering distractors leaves the target as the only item competing for selection; thus solving the binding problem. In the present article we explore the theoretical and empirical basis of this process of active distractor inhibition in search. Specific experiments that provide strong evidence for a process of active distractor inhibition in search are highlighted. In the final part of the article we consider how distractor inhibition, as defined here, may be realized at a neurophysiological level (Treisman and Sato, 1990). PMID:22908002

Specific binding of nicergoline on an alpha1-like adrenoreceptor in the rat retina.

PubMed

Lograno, M D; Tricarico, D; Masciopinto, V; Scuderl, A C

2000-02-01

Systemic treatment with nicergoline, an ergoline derivative showing alpha1-antagonist properties, causes vasodilatation in the eye without apparent untoward cardiovascular effects. In the present work we investigated the ability of nicergoline to inhibit the binding of radiolabelled prazosin in the rat retina and cortex. We found that nicergoline inhibited [3H]prazosin binding in both tissues, being more potent than unlabelled prazosin in the retinal tissue. The competition curves of the ergoline derivative were well fitted by a one-site model in the cortical tissue, with an IC50 (concentration of the drugs needed to inhibit the binding of labelled prazosin by 50%) of 2.54 x 10(-8) M, and by a two-site model in the retinal tissue, with IC50 values of 7.08 x 10(-12) M and 1.82 x 10(-5) M. 2-(2,6 dimetoxyphenoxyethyl) aminomethyl-1,4-benzodioxane hydrochloride (WB4101) and phentolamine, selective ligands for the high-affinity binding site for prazosin, in particular the alpha1A-site, fully inhibited prazosin binding in the cortex but only partially inhibited prazosin binding in the retina, being less potent in this tissue than either nicergoline or prazosin. Our results suggest that a binding component of alpha1-adrenoreceptors is expressed to a lesser extent in the retina than the cortex, leading to a reduced response of the retinal tissue to prazosin, and more particularly to WB4101 and phentolamine. The selective binding of the nicergoline on this retinal adrenoreceptor may explain the peculiar efficacy of the drug in ocular pathophysiology.

Molecular mechanisms of immunosuppression by cyclosporins.

PubMed

Zenke, G; Baumann, G; Wenger, R; Hiestand, P; Quesniaux, V; Andersen, E; Schreier, M H

1993-06-23

Despite the successful clinical application of the immunosuppressive drug cyclosporin A (CsA, Sandimmun), its precise mechanism of action in the process of T cell activation remains elusive. CsA binds to the high-affinity cytosolic receptor cyclophilin whose peptidyl-prolyl cis-trans isomerase activity is inhibited upon binding. The linkage of this effect with the inhibition of the T cell receptor-mediated signal transduction pathway, which leads to a suppression of lymphokine gene transcription, is still unclear. We analyzed the relationship between cyclophilin-binding and immunosuppressive activity (e.g., effect on IL-2 transcription) of cyclosporin derivatives in vitro. The results show that binding to cyclophilin is required, but not sufficient for immunosuppression. Cyclosporin analogues which completely lack immunosuppressive activity but fully retained their cyclophilin-binding capacity antagonize the immunosuppressive activity of CsA. These derivatives inhibit the isomerase activity of cyclophilin, which clearly demonstrates that inhibition of the cyclophilin isomerase activity does not lead to immunosuppression. In analogy to the other immunosuppressants of microbial origin, FK-506 and rapamycin, a specific structure of the "effector" domain of CsA, which is unrelated to the cyclophilin-binding domain, determines the biological activity. In the nucleus, CsA interferes with the DNA-binding of inducible transcription factors to their respective DNA motifs within lymphokine promoters by affecting intracellular translocation of transcription factor subunits.

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

PubMed Central

Robinson, Amanda R.; Kwek, Swee Sen; Kenney, Shannon C.

2012-01-01

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells. PMID:22346751

Isolation and characterization of a specific receptor for human albumin on a group L Streptococcus.

PubMed

Lämmler, C

1988-08-01

Certain group L streptococci demonstrate surface receptors for human albumin. Binding of 125I-albumin to group L streptococci could be inhibited by unlabelled albumin preparations from humans, dogs, mice and bovines, but not by albumin from rabbits. The albumin-binding proteins (ABP) could be solubilized from the streptococcal surface by hot acid treatment of the bacteria and isolated by affinity chromatography on human-albumin sepharose. ABP and specific antisera produced against ABP inhibited 125I-albumin binding to group L streptococci. The molecular weight of ABP determined by SDS-PAGE and Western blotting, was approximately 48,000 Dalton. ABP preparations of group G streptococci isolated from bovines and humans demonstrated cross reactivity with antiserum produced against group L streptococcal ABP.

Functional analysis of CedA based on its structure: residues important in binding of DNA and RNA polymerase and in the cell division regulation.

PubMed

Abe, Yoshito; Fujisaki, Naoki; Miyoshi, Takanori; Watanabe, Noriko; Katayama, Tsutomu; Ueda, Tadashi

2016-02-01

DnaAcos, a mutant of the initiator DnaA, causes overinitiation of chromosome replication in Escherichia coli, resulting in inhibition of cell division. CedA was found to be a multi-copy suppressor which represses the dnaAcos inhibition of cell division. However, functional mechanism of CedA remains elusive except for previously indicated possibilities in binding to DNA and RNA polymerase. In this study, we searched for the specific sites of CedA in binding of DNA and RNA polymerase and in repression of cell division inhibition. First, DNA sequence to which CedA preferentially binds was determined. Next, the several residues and β4 region in CedA C-terminal domain was suggested to specifically interact with the DNA. Moreover, we found that the flexible N-terminal region was required for tight binding to longer DNA as well as interaction with RNA polymerase. Based on these results, several cedA mutants were examined in ability for repressing dnaAcos cell division inhibition. We found that the N-terminal region was dispensable and that Glu32 in the C-terminal domain was required for the repression. These results suggest that CedA has multiple roles and residues with different functions are positioned in the two regions. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter.

PubMed

Sheridan, P L; Schorpp, M; Voz, M L; Jones, K A

1995-03-03

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

Characterization of melatonin binding sites in the Harderian gland and median eminence of the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez-Gonzalez, M.A.; Calvo, J.R.; Rubio, A.

The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using ({sup 125}I)melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37{degree}C after 30-60 min incubation. Binding was also dependent on protein concentration. The specific binding of ({sup 125}I)melatonin was saturable, exhibiting only the class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8more » fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of ({sup 125}I)melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the ({sup 125}I)melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland.« less

Inhibition of Catalase by Tea Catechins in Free and Cellular State: A Biophysical Approach

PubMed Central

Pal, Sandip; Dey, Subrata Kumar; Saha, Chabita

2014-01-01

Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) with catalase were observed to be 2.27×106 M−1 and 1.66×106 M−1, respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC50 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition. PMID:25025898

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

PubMed

Szelag, Malgorzata; Sikorski, Krzysztof; Czerwoniec, Anna; Szatkowska, Katarzyna; Wesoly, Joanna; Bluyssen, Hans A R

2013-11-15

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors. © 2013 Elsevier B.V. All rights reserved.

Autoinhibition of Munc18-1 modulates synaptobrevin binding and helps to enable Munc13-dependent regulation of membrane fusion.

PubMed

Sitarska, Ewa; Xu, Junjie; Park, Seungmee; Liu, Xiaoxia; Quade, Bradley; Stepien, Karolina; Sugita, Kyoko; Brautigam, Chad A; Sugita, Shuzo; Rizo, Josep

2017-05-06

Munc18-1 orchestrates SNARE complex assembly together with Munc13-1 to mediate neurotransmitter release. Munc18-1 binds to synaptobrevin, but the relevance of this interaction and its relation to Munc13 function are unclear. NMR experiments now show that Munc18-1 binds specifically and non-specifically to synaptobrevin. Specific binding is inhibited by a L348R mutation in Munc18-1 and enhanced by a D326K mutation designed to disrupt the 'furled conformation' of a Munc18-1 loop. Correspondingly, the activity of Munc18-1 in reconstitution assays that require Munc18-1 and Munc13-1 for membrane fusion is stimulated by the D326K mutation and inhibited by the L348R mutation. Moreover, the D326K mutation allows Munc13-1-independent fusion and leads to a gain-of-function in rescue experiments in Caenorhabditis elegans unc-18 nulls. Together with previous studies, our data support a model whereby Munc18-1 acts as a template for SNARE complex assembly, and autoinhibition of synaptobrevin binding contributes to enabling regulation of neurotransmitter release by Munc13-1.

Specific receptor for inositol-1,4,5-trisphosphate in permeabilized rabbit neutrophils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradford, P.G.; Spat, A.; Rubin, R.P.

1986-03-05

Neutrophil chemotaxis and degranulation are resultant, in part, from the mobilization of intracellular calcium by inositol-1,4,5-trisphosphate ((1,4,5)IP/sub 3/), one of the products of chemoattractant-stimulated phospholipase C activity. High specific activity (ca. 40 Ci/mmol) (/sup 32/P)(1,4,5)IP/sub 3/ was prepared from (..gamma..-/sup 32/P)ATP-labeled human erythrocyte ghosts and was used in binding assays with saponin-permeabilized rabbit peritoneal neutrophils. At 4/sup 0/C and in the presence of inhibitors of the IP/sub 3/ 5-phosphomonoesterase, (/sup 32/P)(1,4,5)IP/sub 3/ rapidly associated with a specific binding component which saturated within 60s. Nonspecific binding, taken as the residual binding in the presence of 10 ..mu..M (1,4,5)IP/sub 3/, was 15%more » of the total. No specific binding was detected using intact cells. The specific binding to permeable cells was reversible (t/sup 1/2/ approx. 60s) and could be inhibited in a dose-dependent manner by (1,4,5)IP/sub 3/ (EC/sub 50/ = 30 nM) and by other calcium mobilizing inositol phosphates ((2,4,5)IP/sub 3/) but not by inactive analogs ((1,4)IP/sub 2/, (4,5)IP/sub 2/, (1)IP). The dose-responses of (1,4,5)IP/sub 3/ and (2,4,5)IP/sub 3/ in inhibiting (/sup 32/P)(1,4,5)IP/sub 3/ specific binding correlated well with their abilities to release Ca/sup 2 +/ from nonmitochondrial vesicular stores in the same preparation of cells, suggesting that the authors have identified the physiological receptor for (1,4,5)IP/sub 3/.« less

Autoradiographic localization of endothelin-1 binding sites in porcine skin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Y.D.; Springall, D.R.; Wharton, J.

Autoradiographic techniques and {sup 125}I-labeled endothelin-1 were used to study the distribution of endothelin-1 binding sites in porcine skin. Specific endothelin-1 binding sites were localized to blood vessels (capillaries, deep cutaneous vascular plexus, arteries, and arterioles), the deep dermal and connective tissue sheath of hair follicles, sebaceous and sweat glands, and arrector pili muscle. Specific binding was inhibited by endothelin-2 and endothelin-3 as well as endothelin-1. Non-specific binding was found in the epidermis and the medulla of hair follicles. No binding was found in connective tissue or fat. These vascular binding sites may represent endothelin receptors, in keeping with themore » known cutaneous vasoconstrictor actions of the peptide. If all binding sites are receptors, the results suggest that endothelin could also regulate the function of sweat glands and may have trophic effects in the skin.« less

Molecular mechanisms of DNA repair inhibition by caffeine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Selby, C.P.; Sancar, A.

1990-05-01

Caffeine potentiates the mutagenic and lethal effects of genotoxic agents. It is thought that this is due, at least in some organisms, to inhibition of DNA repair. However, direct evidence for inhibition of repair enzymes has been lacking. Using purified Escherichia coli DNA photolyase and (A)BC excinuclease, we show that the drug inhibits photoreactivation and nucleotide excision repair by two different mechanisms. Caffeine inhibits photoreactivation by interfering with the specific binding of photolyase to damaged DNA, and it inhibits nucleotide excision repair by promoting nonspecific binding of the damage-recognition subunit, UvrA, of (A)BC excinuclease. A number of other intercalators, includingmore » acriflavin and ethidium bromide, appear to inhibit the excinuclease by a similar mechanism--that is, by trapping the UvrA subunit in nonproductive complexes on undamaged DNA.« less

Mechanism of lignin inhibition of enzymatic biomass deconstruction

DOE PAGES

Vermaas, Josh V.; Petridis, Loukas; Qi, Xianghong; ...

2015-12-01

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process. By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose bindingmore » of TrCel7A (Y466, Y492, and Y493). In conclusion, lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.« less

Interaction of berberine with human platelet. alpha. sub 2 adrenoceptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hui, Ka Kit; Yu, Jun Liang; Chan, Wai Fong A.

1991-01-01

Berberine was found to inhibit competitively the specific binding of ({sup 3}H)-yohimbine. The displacement curve was parallel to those of clonidine, epinephrine, norepinephrine, with the rank order of potency (IC{sub 50}) being clonidine {gt} epinephrine {gt} norepinephrine (14.5 {mu}M) = berberine. Increasing concentrations of berberine from 0.1 {mu}M to 10 {mu}M inhibited ({sup 3}H)-yohimbine binding, shifting the saturation binding curve to the right without decreasing the maximum binding capacity. In platelet cyclic AMP accumulation experiments, berberine at concentrations of 0.1 {mu}M to 0.1 mM inhibited the cAMP accumulation induced by 10 {mu}M prostaglandin E{sub 1} in a dose dependent manner,more » acting as an {alpha}{sub 2} adrenoceptor agonist. In the presence of L-epinephrine, berberine blocked the inhibitory effect of L-epinephrine behaving as an {alpha}{sub 2} adrenoceptor antagonist.« less

Binding of extracellular matrix proteins to Aspergillus fumigatus conidia.

PubMed Central

Gil, M L; Peñalver, M C; Lopez-Ribot, J L; O'Connor, J E; Martinez, J P

1996-01-01

As detected by confocal immunofluorescence microscopy, binding of fibronectin and laminin appeared to be associated with the protrusions present on the outer cell wall layer of resting Aspergillus fumigatus conidia. Flow cytometry confirmed that binding of laminin to conidia was dose dependent and saturable. Laminin binding was virtually eliminated in trypsin-treated organisms, thus suggesting the protein nature of the binding site. Conidia were also able to specifically adhere to laminin immobilized on microtiter plates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) with laminin and antilaminin antibody of whole conidial homogenates allowed identification, among the complex array of protein and glycoprotein species, of one polypeptide with an apparent molecular mass of 37 kDa which specifically interacts with laminin. The fact that binding of conidia to soluble or immobilized laminin or fibronectin was inhibited by fibronectin or laminin, respectively, suggests the existence of common binding sites for both ligands on the surface of conidia. Intact conidia were also able to adhere to type I and IV collagen immobilized on microtiter plates; adhesion was found to be dose dependent and saturable. Adhesion to immobilized type I and IV collagen was markedly inhibited by laminin and weakly inhibited by fibronectin. Coincubation of conidia with Arg-Gly-Asp (RGD) peptides caused a dose-dependent decrease in binding of cells to immobilized or soluble fibronectin, yet interaction of cells with soluble or immobilized laminin and type I and IV collagen remained unaffected. Interactions described here could be important in mediating attachment of the fungus to host tissues, thus playing a role in the establishment of the disease. PMID:8945572

SERS and MD simulation studies of a kinase inhibitor demonstrate the emergence of a potential drug discovery tool.

PubMed

Karthigeyan, Dhanasekaran; Siddhanta, Soumik; Kishore, Annavarapu Hari; Perumal, Sathya S R R; Ågren, Hans; Sudevan, Surabhi; Bhat, Akshay V; Balasubramanyam, Karanam; Subbegowda, Rangappa Kanchugarakoppal; Kundu, Tapas K; Narayana, Chandrabhas

2014-07-22

We demonstrate the use of surface-enhanced Raman spectroscopy (SERS) as an excellent tool for identifying the binding site of small molecules on a therapeutically important protein. As an example, we show the specific binding of the common antihypertension drug felodipine to the oncogenic Aurora A kinase protein via hydrogen bonding interactions with Tyr-212 residue to specifically inhibit its activity. Based on SERS studies, molecular docking, molecular dynamics simulation, biochemical assays, and point mutation-based validation, we demonstrate the surface-binding mode of this molecule in two similar hydrophobic pockets in the Aurora A kinase. These binding pockets comprise the same unique hydrophobic patches that may aid in distinguishing human Aurora A versus human Aurora B kinase in vivo. The application of SERS to identify the specific interactions between small molecules and therapeutically important proteins by differentiating competitive and noncompetitive inhibition demonstrates its ability as a complementary technique. We also present felodipine as a specific inhibitor for oncogenic Aurora A kinase. Felodipine retards the rate of tumor progression in a xenografted nude mice model. This study reveals a potential surface pocket that may be useful for developing small molecules by selectively targeting the Aurora family kinases.

Effects of hexamethonium, phenothiazines, propranolol and ephedrine on acetylcholinesterase carbamylation by physostigmine, aldicarb and carbaryl: interaction between the active site and the functionally distinct peripheral sites in acetylcholinesterase.

PubMed

Singh, A K; Spassova, D

1998-01-01

Physostigmine, aldicarb and carbaryl were potent inhibitors of acetylcholinesterase (AChE). The physostigmine-inhibited AChE fluoresced at 300 nm excitation and 500 nm emission wavelengths, but the aldicarb and carbaryl inhibited enzyme did not. This suggests that the carbamylated active center is not the fluorescing site in AChE. The fluorescence intensity of physostigmine-inhibited AChE decreased with increasing the substrate (acetylthiocholine) concentration, thus indicating that physostigmine binding to the active site is essential for the development of fluorescence. Thus, the physostigmine-inhibited AChE fluoresces due to the binding of trimethylpyrrolo[2,3-b]indol (TMPI) moiety, formed by the hydrolysis of physostigmine, to a peripheral site in AChE. The fluorescence intensity of the physostigmine-inhibited enzyme decreased when the inhibited-enzyme was dialyzed for either 30 min that poorly reactivated the enzyme or 180 min that fully reactivated the enzyme. This suggests that dialysis dissociates the AChE-TMPI complex much faster than it reactivates the carbamylated AChE. Ephedrine, propranolol and phenothiazines including trifluoparazine (TPZ) caused non-competitive inhibition, while hexamethonium caused an uncompetitive inhibition of AChE activity. TPZ, upon binding with AChE, formed a fluorescent TPZ-enzyme complex. The fluorescence intensity of TPZ-AChE complex was effectively decreased by ephedrine, but not by propranolol or hexamethonium. This indicates that TPZ and ephedrine bind to the same site in AChE which is different from the site/or sites to which propranolol or hexamethonium bind. Hexamethonium protected AChE from inhibition by carbamates and decreased the fluorescence intensity of the physostigmine-inhibited AChE. Phenothiazines and ephedrine did not modulate the enzyme inhibition or the fluorescence intensity of the physostigmine-inhibited AChE. Propranolol and TPZ potentiated the enzyme inhibition and increased the fluorescence intensity in the presence of physostigmine. These compounds, however, did not affect the inhibition of AChE by carbaryl or aldicarb. Ephedrine blocked the effects of TPZ, but did not alter the effects of propranolol on physostigmine-inhibited AChE. AChE, therefore, contains multiple peripheral binding sites which, upon binding to specific ligands, transduce differential signals to the active center.

Human milk containing specific secretory IgA inhibits binding of Giardia lamblia to nylon and glass surfaces.

PubMed

Samra, H K; Ganguly, N K; Mahajan, R C

1991-06-01

The effects of human milk, containing specific secretory IgA, on the adherence of Giardia lamblia trophozoites in the presence and in the absence of intestinal mucus in vitro were studied. It was found that the trophozoites treated with breast milk, containing specific secretory IgA to G. lamblia, showed a significant decrease (p less than 0.01) in adherence to nylon fibre columns and glass surfaces than did trophozoites treated with milk containing no SIgA antibodies. The adherence to glass surfaces was significantly more (p less than 0.01) in the presence of intestinal mucus than when the mucus was absent. Milk that did not contain specific secretory SIgA to G. lamblia did not decrease the adherence to glass surfaces either in the presence or in the absence of mucus. The fluorescence study revealed the binding of specific secretory IgA on the trophozoite surface. The results suggest that binding of SIgA antibodies in milk to G. lamblia trophozoites inhibits parasite adherence, thus protecting against this infection in breast-fed babies.

Structural basis for the inhibition of voltage-dependent K+ channel by gating modifier toxin

PubMed Central

Ozawa, Shin-ichiro; Kimura, Tomomi; Nozaki, Tomohiro; Harada, Hitomi; Shimada, Ichio; Osawa, Masanori

2015-01-01

Voltage-dependent K+ (Kv) channels play crucial roles in nerve and muscle action potentials. Voltage-sensing domains (VSDs) of Kv channels sense changes in the transmembrane potential, regulating the K+-permeability across the membrane. Gating modifier toxins, which have been used for the functional analyses of Kv channels, inhibit Kv channels by binding to VSD. However, the structural basis for the inhibition remains elusive. Here, fluorescence and NMR analyses of the interaction between VSD derived from KvAP channel and its gating modifier toxin, VSTx1, indicate that VSTx1 recognizes VSD under depolarized condition. We identified the VSD-binding residues of VSTx1 and their proximal residues of VSD by the cross-saturation (CS) and amino acid selective CS experiments, which enabled to build a docking model of the complex. These results provide structural basis for the specific binding and inhibition of Kv channels by gating modifier toxins. PMID:26382304

The biological function of an insect antifreeze protein simulated by molecular dynamics

PubMed Central

Kuiper, Michael J; Morton, Craig J; Abraham, Sneha E; Gray-Weale, Angus

2015-01-01

Antifreeze proteins (AFPs) protect certain cold-adapted organisms from freezing to death by selectively adsorbing to internal ice crystals and inhibiting ice propagation. The molecular details of AFP adsorption-inhibition is uncertain but is proposed to involve the Gibbs–Thomson effect. Here we show by using unbiased molecular dynamics simulations a protein structure-function mechanism for the spruce budworm Choristoneura fumiferana AFP, including stereo-specific binding and consequential melting and freezing inhibition. The protein binds indirectly to the prism ice face through a linear array of ordered water molecules that are structurally distinct from the ice. Mutation of the ice binding surface disrupts water-ordering and abolishes activity. The adsorption is virtually irreversible, and we confirm the ice growth inhibition is consistent with the Gibbs–Thomson law. DOI: http://dx.doi.org/10.7554/eLife.05142.001 PMID:25951514

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

PubMed

Kohlmann, Anna; Zech, Stephan G; Li, Feng; Zhou, Tianjun; Squillace, Rachel M; Commodore, Lois; Greenfield, Matthew T; Lu, Xiaohui; Miller, David P; Huang, Wei-Sheng; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Zhang, Sen; Dodd, Rory; Liu, Shuangying; Xu, Rongsong; Xu, Yongjin; Miret, Juan J; Rivera, Victor; Clackson, Tim; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2013-02-14

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

The immunosuppressives FK 506 and cyclosporin A inhibit the generation of protein factors binding to the two purine boxes of the interleukin 2 enhancer.

PubMed Central

Brabletz, T; Pietrowski, I; Serfling, E

1991-01-01

Like Cyclosporin A (CsA), the macrolide FK 506 is a potent immunosuppressive that inhibits early steps of T cell activation, including the synthesis of Interleukin 2 (II-2) and numerous other lymphokines. The block of II-2 synthesis occurs at the transcriptional level. At concentrations that block T cell activation, FK 506 and CsA inhibit the proto-enhancer activity of Purine boxes of the II-2 promoter and the generation of lymphocyte-specific factors binding to the Purine boxes. Under the same conditions, the DNA binding of other II-2 enhancer factors remains unaffected by both compounds. These results support the view that FK 506 and CsA, which both inhibit the activity of peptidylprolyl cis/trans isomerases, suppress T cell activation by a similar, if not identical mechanism. Images PMID:1707162

The immunosuppressives FK 506 and cyclosporin A inhibit the generation of protein factors binding to the two purine boxes of the interleukin 2 enhancer.

PubMed

Brabletz, T; Pietrowski, I; Serfling, E

1991-01-11

Like Cyclosporin A (CsA), the macrolide FK 506 is a potent immunosuppressive that inhibits early steps of T cell activation, including the synthesis of Interleukin 2 (II-2) and numerous other lymphokines. The block of II-2 synthesis occurs at the transcriptional level. At concentrations that block T cell activation, FK 506 and CsA inhibit the proto-enhancer activity of Purine boxes of the II-2 promoter and the generation of lymphocyte-specific factors binding to the Purine boxes. Under the same conditions, the DNA binding of other II-2 enhancer factors remains unaffected by both compounds. These results support the view that FK 506 and CsA, which both inhibit the activity of peptidylprolyl cis/trans isomerases, suppress T cell activation by a similar, if not identical mechanism.

GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules

PubMed Central

Hur, Eun-Mi; Saijilafu; Lee, Byoung Dae; Kim, Seong-Jin; Xu, Wen-Lin; Zhou, Feng-Quan

2011-01-01

Suppression of glycogen synthase kinase 3 (GSK3) activity in neurons yields pleiotropic outcomes, causing both axon growth promotion and inhibition. Previous studies have suggested that specific GSK3 substrates, such as adenomatous polyposis coli (APC) and collapsin response mediator protein 2 (CRMP2), support axon growth by regulating the stability of axonal microtubules (MTs), but the substrate(s) and mechanisms conveying axon growth inhibition remain elusive. Here we show that CLIP (cytoplasmic linker protein)-associated protein (CLASP), originally identified as a MT plus end-binding protein, displays both plus end-binding and lattice-binding activities in nerve growth cones, and reveal that the two MT-binding activities regulate axon growth in an opposing manner: The lattice-binding activity mediates axon growth inhibition induced by suppression of GSK3 activity via preventing MT protrusion into the growth cone periphery, whereas the plus end-binding property supports axon extension via stabilizing the growing ends of axonal MTs. We propose a model in which CLASP transduces GSK3 activity levels to differentially control axon growth by coordinating the stability and configuration of growth cone MTs. PMID:21937714

Monoclonal and anti-idiotypic anti-EBV/C3d receptor antibodies detect two binding sites, one for EBV and one for C3d on glycoprotein 140, the EBV/C3dR, expressed on human B lymphocytes.

PubMed

Barel, M; Fiandino, A; Delcayre, A X; Lyamani, F; Frade, R

1988-09-01

Glycoprotein (gp) 140, the EBV/C3dR of B lymphocytes, is a membrane site involved in human cell regulation. To analyze the specificities of the binding sites for EBV and for C3d on the gp 140 molecule, two distinct approaches were used. First, anti-EBV/C3dR mAb were prepared against highly purified EBV/C3dR. Nine anti-EBV/C3dR mAb were obtained. Four of these anti-EBV/C3dR mAb inhibited C3d binding but not EBV binding on gp 140, whereas four others exerted an inverse effect. These differences could not be due to differences in isotype, antibody concentration, affinity constant, and number of molecules bound on cell surface, as these parameters were identical for the nine used mAb. Second, polyclonal anti-idiotypic antibodies (Ab2) were prepared against F(ab)'2 fragments of polyclonal anti-EBV/C3dR (Ab1). Ab2 recognized the variable portion of Ab1 as controlled by immunoblotting experiments. Ab2, which did not react with the cell surface, inhibited Ab1 binding on Raji cells. Ab2 mimicked the EBV/C3dR by its properties to bind to particle-bound C3d and EBV, preventing their binding on Raji cell surface. C3d binding specificities contained in Ab2 were isolated by affinity chromatography on C3b/C3bi-Sepharose. These specificities, being the internal image of C3d binding site of EBV/C3dR, reacted with Ab1 and inhibited particle-bound C3d binding on Raji cells but did not react with EBV. Taken together, these data support strongly that gp 140, the EBV/C3dR, carried two distinct binding sites, one for EBV and one for C3d.

Interaction of tachykinins with their receptors studied with cyclic analogues of substance P and neurokinin B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ploux, O.; Lavielle, S.; Chassaing, G.

1987-11-01

The activities of two groups of cyclic agonists of substance P (SP) have been studied. The disulfide bridge constraints have been designed on the basis of conformational studies on SP and physalaemin indicating an ..cap alpha..-helical structure for the core of these two tachykinins (group I) and a folding of the C-terminal carboxamide towards the side chains of the glutamines 5 and 6 (group II). Only peptides simulating the ..cap alpha..-helix present substantial potencies. (Cys/sup 3,6/)SP is as active as SP in inhibiting /sup 125/I-labeled Bolton and Hunter SP-specific binding on rat brain synaptosomes and on dog carotid bioassay, twomore » assays specific for the neurokinin 1 receptor. Moreover, (Cys/sup 3,6/)SP is a potent as neurokinin B in inhibiting /sup 125/I-labeled Bolton and Hunter eledoisin-specific binding on rat cortical synaptosomes as well as in stimulating rat portal vein, two tests specific for the neurokinin 3 receptor. Interestingly, in contrast to neurokinin B, (Cys/sup 3,6/)SP is a weak agonist of the neurokinin 2 receptor subtype, as evidenced by its binding potency in inhibiting /sup 3/H-labeled neurokinin A-specific binding on rat duodenum and in inducing the contractions of the rabbit pulmonary artery, a neurokinin 2-type bioassay. To increase the specificity of the cyclic analogue (Cys/sup 3,6/)SP positions 8 and 9 were modified. Collectively, these results suggest that the neurokinin 1 and neurokinin 3 tachykinin receptors may recognize a similar three-dimensional structure of the core of the tachykinins. Different orientations of the common C-terminal tripeptide may be related to the selectivity for the different receptor subtypes.« less

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation.

PubMed

Lekowski, R; Collard, C D; Reenstra, W R; Stahl, G L

2001-02-01

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O(2), 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 +/- 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (< or = 100 micromol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC(50) = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC(50) approximately 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress.

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation

PubMed Central

Lekowski, Robert; Collard, Charles D.; Reenstra, Wende R.; Stahl, Gregory L.

2001-01-01

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O2, 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 ± 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (≤ 100 μmol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC50 = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC50 ≈ 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress. PMID:11266613

Mutant botrocetin-2 inhibits von Willebrand factor-induced platelet agglutination.

PubMed

Matsui, T; Hori, A; Hamako, J; Matsushita, F; Ozeki, Y; Sakurai, Y; Hayakawa, M; Matsumoto, M; Fujimura, Y

2017-03-01

Essentials Botrocetin-2 (Bot2) binds to von Willebrand factor (VWF) and induces platelet agglutination. We identified Bot2 residues that are required for binding to VWF and glycoprotein (GP) Ib. We produced a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Mutant Bot2 could be used as a potential anti-thrombotic reagent to block VWF-GPIb interaction. Background Botrocetin-2 (Bot2) is a botrocetin-like protein composed of α and β subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination. Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-botrocetin column. The mutant rBot2 with either Ala at Asp70 in the β subunit (Aspβ70Ala), or Argβ115Ala and Lysβ117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Aspβ70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Argβ115Ala/Lysβ117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Argβ115 and Lysβ117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions. Conclusions Asp70 in the β subunit of botrocetin is important for VWF binding, and Arg115 and Lys117 in the β subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Argβ115Glu and Lysβ117Glu, repels GPIb and might have potential as an antithrombotic reagent that specifically blocks VWF function. This is the first report on an artificial botrocetin that can inhibit the VWF-GPIb interaction. © 2017 International Society on Thrombosis and Haemostasis.

A functionalized poly(ethylene glycol)-based bioassay surface chemistry that facilitates bio-immobilization and inhibits non-specific protein, bacterial, and mammalian cell adhesion

PubMed Central

Harbers, Gregory M.; Emoto, Kazunori; Greef, Charles; Metzger, Steven W.; Woodward, Heather N.; Mascali, James J.; Grainger, David W.; Lochhead, Michael J.

2008-01-01

This paper describes a new bioassay surface chemistry that effectively inhibits non-specific biomolecular and cell binding interactions, while providing a capacity for specific immobilization of desired biomolecules. Poly(ethylene glycol) (PEG) as the primary component in nonfouling film chemistry is well-established, but the multicomponent formulation described here is unique in that it (1) is applied in a single, reproducible, solution-based coating step; (2) can be applied to diverse substrate materials without the use of special primers; and (3) is readily functionalized to provide specific attachment chemistries. Surface analysis data are presented, detailing surface roughness, polymer film thickness, and film chemistry. Protein non-specific binding assays demonstrate significant inhibition of serum, fibrinogen, and lysozyme adsorption to coated glass, indium tin oxide, and tissue culture polystyrene dishes. Inhibition of S. aureus and K. pneumoniae microbial adhesion in a microfluidic flow cell, and inhibition of fibroblast cell adhesion from serum-based cell culture is shown. Effective functionalization of the coating is demonstrated by directing fibroblast adhesion to polymer surfaces activated with an RGD peptide. Batch-to-batch reproducibility data are included. The in situ cross-linked PEG-based coating chemistry is unique in its formulation, and its surface properties are attractive for a broad range of in vitro bioassay applications. PMID:18815622

Functional characterization of chitin-binding lectin from Solanum integrifolium containing anti-fungal and insecticidal activities.

PubMed

Chen, Chang-Shan; Chen, Chun-Yi; Ravinath, Divya Malathy; Bungahot, Agustina; Cheng, Chi-Ping; You, Ren-In

2018-01-03

Along with the rapid development of glycomic tools, the study of lectin-carbohydrate interactions has expanded, opening the way for applications in the fields of analytic, diagnostic, and drug delivery. Chitin-binding lectins (CBLs) play roles in immune defense against chitin-containing pathogens. CBLs from species of the Solanaceae family, such as tomato, potato and jimsonweed, display different binding specificities to sugar chains containing poly-N-acetyllactosamine. In this report, CBLs from Solanum integrifolium were isolated by ion exchange chromatography. The fractions showed hemagglutination activity (HA). The recombinant CBL in the 293F cell culture supernatant was able to inhibit the growth of Rhizoctonia solani and Colletotrichum gloeosporioide. Furthermore, the carbohydrate-binding property of CBLs was confirmed with the inhibition of HA. Binding of CBL to Spodoptera frugiperda (sf21) insect cells can partly be inhibited by N-Acetylglucosamine (GlcNAc), which is related to decrease mitochondrial membrane potential of sf21 cells. The results showed that CBL exhibited antifungal properties and inhibited insect cell growth, which is directly correlated to the lectin-carbohydrate interaction. Further identification and characterization of CBLs will help to broaden their scope of application in plant defense and in biomedical applications.

Analysis of surface properties of fixed and live cells using derivatized agarose beads.

PubMed

Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

2002-01-01

A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

Binding of CD40L to Mac-1’s I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis – but does not affect immunity and thrombosis in mice

PubMed Central

Wolf, Dennis; Hohmann, Jan-David; Wiedemann, Ansgar; Bledzka, Kamila; Blankenbach, Hermann; Marchini, Timoteo; Gutte, Katharina; Zeschky, Katharina; Bassler, Nicole; Hoppe, Natalie; Rodriguez, Alexandra Ortiz; Herr, Nadine; Hilgendorf, Ingo; Stachon, Peter; Willecke, Florian; Dürschmied, Daniel; von zur Mühlen, Constantin; Soloviev, Dmitry A.; Zhang, Li; Bode, Christoph; Plow, Edward F.; Libby, Peter; Peter, Karlheinz; Zirlik, Andreas

2012-01-01

Rationale CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and haemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Objective Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. Methods and Results CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. cM7, a cyclisized version optimized for in vivo use, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr-/- mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. Conclusions We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L. PMID:21998326

Identification of 50- and 23-/25-kDa HeLa cell membrane glycoproteins involved in poliovirus infection: occurrence of poliovirus specific binding sites on susceptible and nonsusceptible cells.

PubMed

Barnert, R H; Zeichhardt, H; Habermehl, K O

1992-02-01

Glycoproteins in the range 50 and 23/25 kDa were identified as poliovirus specific binding sites on HeLa cells with the monoclonal antibody mAb 122. mAb 122 is characterized by its partial inhibiting effect on poliovirus reproduction and adsorption when prebound to HeLa cells. The binding sites are endocytosed in native cells and specific for poliovirus as mAb 122 did not interfere with the adsorption of human rhinovirus type 14 (HRV 14). The poliovirus binding sites are present also on nonprimate so called nonsusceptible cells, e.g., mouse L-cells, as could be shown with sensitive ELISA based binding assays and performance of binding studies with fixed cells at 37 degrees.

Phosphorylation-dependent mineral-type specificity for apatite-binding peptide sequences.

PubMed

Addison, William N; Miller, Sharon J; Ramaswamy, Janani; Mansouri, Ahmad; Kohn, David H; McKee, Marc D

2010-12-01

Apatite-binding peptides discovered by phage display provide an alternative design method for creating functional biomaterials for bone and tooth tissue repair. A limitation of this approach is the absence of display peptide phosphorylation--a post-translational modification important to mineral-binding proteins. To refine the material specificity of a recently identified apatite-binding peptide, and to determine critical design parameters (net charge, charge distribution, amino acid sequence and composition) controlling peptide affinity for mineral, we investigated the effects of phosphorylation and sequence scrambling on peptide adsorption to four different apatites (bone-like mineral, and three types of apatite containing initially 0, 5.6 and 10.5% carbonate). Phosphorylation of the VTKHLNQISQSY peptide (VTK peptide) led to a 10-fold increase in peptide adsorption (compared to nonphosphorylated peptide) to bone-like mineral, and a 2-fold increase in adsorption to the carbonated apatite, but there was no effect of phosphorylation on peptide affinity to pure hydroxyapatite (without carbonate). Sequence scrambling of the nonphosphorylated VTK peptide enhanced its specificity for the bone-like mineral, but scrambled phosphorylated VTK peptide (pVTK) did not significantly alter mineral-binding suggesting that despite the importance of sequence order and/or charge distribution to mineral-binding, the enhanced binding after phosphorylation exceeds any further enhancement by altered sequence order. Osteoblast culture mineralization was dose-dependently inhibited by pVTK and to a significantly lesser extent by scrambled pVTK, while the nonphosphorylated and scrambled forms had no effect, indicating that inhibition of osteoblast mineralization is dependent on both peptide sequence and charge. Computational modeling of peptide-mineral interactions indicated a favorable change in binding energy upon phosphorylation that was unaffected by scrambling. In conclusion, phosphorylation of serine residues increases peptide specificity for bone-like mineral, whose adsorption is determined primarily by sequence composition and net charge as opposed to sequence order. However, sequence order in addition to net charge modulates the mineralization of osteoblast cultures. The ability of such peptides to inhibit mineralization has potential utility in the management of pathologic calcification. Copyright © 2010 Elsevier Ltd. All rights reserved.

Inhibition and Ultraviolet-Induced Chemical Modification of UDP-Glucose:(1,3)-β-Glucan (Callose) Synthase by Chlorpromazine 1

PubMed Central

Harriman, Robert W.; Shao, Ai-Ping; Wasserman, Bruce P.

1992-01-01

UDP-glucose:(1,3)-β-glucan (callose) synthase (CS) from storage tissue of red beet (Beta vulgaris L.) was strongly inhibited by the phenothiazine drug chlorpromazine (CPZ). In the absence of ultraviolet irradiation, CPZ was a noncompetitive inhibitor with 50% inhibitory concentration values for plasma membrane and solubilized CS of 100 and 90 μm, respectively. Both the Ca2+- and Mg2+- stimulated components of CS activity were affected. CPZ inhibition was partially alleviated at saturating levels of Ca2+, but not Mg2+, suggesting that CPZ interferes with the Ca2+-binding site of CS. Binding experiments with [14C]CPZ, however, showed strong non-specific partitioning of CPZ into the plasma membrane, providing evidence that perturbation of the membrane environment is probably the predominant mode of inhibition. Ultraviolet irradiation at 254 nm markedly enhanced CPZ inhibition, with complete activity loss following exposure to 4 μm CPZ for 2 min. Inhibition followed a pseudo-first order mechanism with at least three CPZ binding sites per CS complex. Under these conditions, [3H]CPZ was covalently incorporated into plasma membrane preparations by a free radical mechanism; however, polypeptide labeling profiles showed labeling to be largely nonspecific, with many polypeptides labeled even at [3H]CPZ levels as low as 1 μm, and with boiled membranes. Although CPZ is one of the most potent known inhibitors of CS, its use as a photolabel will require a homogeneous CS complex or establishment of conditions that protect against the interaction of CPZ with specific binding sites located on various polypeptide components of the CS complex. PMID:16653219

Dual interaction of agmatine with the rat α2D-adrenoceptor: competitive antagonism and allosteric activation

PubMed Central

Molderings, G J; Menzel, S; Kathmann, M; Schlicker, E; Göthert, M

2000-01-01

In segments of rat vena cava preincubated with [3H]-noradrenaline and superfused with physiological salt solution, the influence of agmatine on the electrically evoked [3H]-noradrenaline release, the EP3 prostaglandin receptor-mediated and the α2D-adrenoceptor-mediated inhibition of evoked [3H]-noradrenaline release was investigated. Agmatine (0.1–10 μM) by itself was without effect on evoked [3H]-noradrenaline release. In the presence of 10 μM agmatine, the prostaglandin E2(PGE2)-induced EP3-receptor-mediated inhibition of [3H]-noradrenaline release was not modified, whereas the α2D-adrenoceptor-mediated inhibition of [3H]-noradrenaline release induced by noradrenaline, moxonidine or clonidine was more pronounced than in the absence of agmatine. However, 1 mM agmatine antagonized the moxonidine-induced inhibition of [3H]-noradrenaline release. Agmatine concentration-dependently inhibited the binding of [3H]-clonidine and [3H]-rauwolscine to rat brain cortex membranes (Ki values 6 μM and 12 μM, respectively). In addition, 30 and 100 μM agmatine increased the rate of association and decreased the rate of dissociation of [3H]-clonidine resulting in an increased affinity of the radioligand for the α2D-adrenoceptors. [14C]-agmatine labelled specific binding sites on rat brain cortex membranes. In competition experiments. [14C]-agmatine was inhibited from binding to its specific recognition sites by unlabelled agmatine, but not by rauwolscine and moxonidine. In conclusion, the present data indicate that agmatine both acts as an antagonist at the ligand recognition site of the α2D-adrenoceptor and enhances the effects of α2-adrenoceptor agonists probably by binding to an allosteric binding site of the α2D-adrenoceptor which seems to be labelled by [14C]-agmatine. PMID:10928978

Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

PubMed

Giuntini, Serena; Reason, Donald C; Granoff, Dan M

2011-09-01

Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

Autoinhibitory mechanisms of ERG studied by molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Lu, Yan; Salsbury, Freddie R.

2015-01-01

ERG, an ETS-family transcription factor, acts as a regulator of differentiation of early hematopoietic cells. It contains an autoinhibitory domain, which negatively regulates DNA-binding. The mechanism of autoinhibitory is still illusive. To understand the mechanism, we study the dynamical properties of ERG protein by molecular dynamics simulations. These simulations suggest that DNA binding autoinhibition associates with the internal dynamics of ERG. Specifically, we find that (1), The N-C terminal correlation in the inhibited ERG is larger than that in uninhibited ERG that contributes to the autoinhibition of DNA-binding. (2), DNA-binding changes the property of the N-C terminal correlation from being anti-correlated to correlated, that is, changing the relative direction of the correlated motions and (3), For the Ets-domain specifically, the inhibited and uninhibited forms exhibit essentially the same dynamics, but the binding of the DNA decreases the fluctuation of the Ets-domain. We also find from PCA analysis that the three systems, even with quite different dynamics, do have highly similar free energy surfaces, indicating that they share similar conformations.

Molecular docking and inhibition studies on the interactions of Bacopa monnieri's potent phytochemicals against pathogenic Staphylococcus aureus.

PubMed

Emran, Talha Bin; Rahman, Md Atiar; Uddin, Mir Muhammad Nasir; Dash, Raju; Hossen, Md Firoz; Mohiuddin, Mohammad; Alam, Md Rashadul

2015-04-17

Bacopa monnieri Linn. (Plantaginaceae), a well-known medicinal plant, is widely used in traditional medicine system. It has long been used in gastrointestinal discomfort, skin diseases, epilepsy and analgesia. This research investigated the in vitro antimicrobial activity of Bacopa monnieri leaf extract against Staphylococcus aureus and the interaction of possible compounds involved in this antimicrobial action. Non-edible plant parts were extracted with ethanol and evaporated in vacuo to obtain the crude extract. A zone of inhibition studies and the minimum inhibitory concentration (MIC) of plant extracts were evaluated against clinical isolates by the microbroth dilution method. Docking study was performed to analyze and identify the interactions of possible antimicrobial compounds of Bacopa monnieri in the active site of penicillin binding protein and DNA gyrase through GOLD 4.12 software. A zone of inhibition studies showed significant (p < 0.05) inhibition capacity of different concentrations of Bacopa monnieri's extract against Staphylococcus aureus. The extract also displayed very remarkable minimum inhibitory concentrations (≥16 μg/ml) which was significant compared to that (≥75 μg/ml) of the reference antibiotic against the experimental strain Staphylococcus aureus. Docking studies recommended that luteolin, an existing phytochemical of Bacopa monnieri, has the highest fitness score and more specificity towards the DNA gyrase binding site rather than penicillin binding protein. Bacopa monnieri extract and its compound luteolin have a significant antimicrobial activity against Staphylococcus aureus. Molecular binding interaction of an in silico data demonstrated that luteolin has more specificity towards the DNA gyrase binding site and could be a potent antimicrobial compound.

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy.

PubMed

Bizzarri, Anna Rita; Santini, Simona; Coppari, Emilia; Bucciantini, Monica; Di Agostino, Silvia; Yamada, Tohru; Beattie, Craig W; Cannistraro, Salvatore

2011-01-01

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53.

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy

PubMed Central

Bizzarri, Anna Rita; Santini, Simona; Coppari, Emilia; Bucciantini, Monica; Di Agostino, Silvia; Yamada, Tohru; Beattie, Craig W; Cannistraro, Salvatore

2011-01-01

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53. PMID:22162658

Lectin-Based Assay for Glycoform-Specific Detection of α2,6-sialylated Transferrin and Carcinoembryonic Antigen in Tissue and Body Fluid.

PubMed

Ito, Hiromi; Hoshi, Kyoka; Honda, Takashi; Hashimoto, Yasuhiro

2018-05-30

Antibodies are useful for detecting glycoprotein antigens, but a conventional antibody recognizes only a protein epitope rather than a glycan. Thus, glycan isoform detection generally requires time- and labor-consuming processes such as lectin affinity column chromatography followed by sandwich ELISA. We recently found antigen-antibody reactions that were inhibited by lectin binding to glycans on the glycoprotein antigen, leading to a convenient glycoform-specific assay. Indeed, Sambucus sieboldiana agglutinin (SSA) lectin, a binder to sialylα2,6galactose residue, inhibited antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform-specific. SSA inhibition was also applicable for visualizing localization of α2,6-sialylated-Tf in a liver section. This is the first immunohistochemical demonstration of glycoform localization in a tissue section. SSA inhibition was utilized for establishing ELISA to quantify α2,6-sialylated carcinoembryonic antigen (CEA), a marker for various cancers. In addition, α2,6-sialylated-CEA was visualized in a colonic adenocarcinoma section by SSA inhibition. The method would further be applicable to a simple and rapid estimation of other α2,6-sialylated glycoproteins and have a potential aid to histopathological diagnosis.

The mechanism of interactions between tea polyphenols and porcine pancreatic alpha‐amylase: Analysis by inhibition kinetics, fluorescence quenching, differential scanning calorimetry and isothermal titration calorimetry

PubMed Central

Sun, Lijun; Gidley, Michael J.

2017-01-01

Scope This study aims to use a combination of biochemical and biophysical methods to derive greater mechanistic understanding of the interactions between tea polyphenols and porcine pancreatic α‐amylase (PPA). Methods and results The interaction mechanism was studied through fluorescence quenching (FQ), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) and compared with inhibition kinetics. The results showed that a higher quenching effect of polyphenols corresponded to a stronger inhibitory activity against PPA. The red‐shift of maximum emission wavelength of PPA bound with some polyphenols indicated a potential structural unfolding of PPA. This was also suggested by the decreased thermostability of PPA with these polyphenols in DSC thermograms. Through thermodynamic binding analysis of ITC and inhibition kinetics, the equilibrium of competitive inhibition was shown to result from the binding of particularly galloylated polyphenols with specific sites on PPA. There were positive linear correlations between the reciprocal of competitive inhibition constant (1/K ic), quenching constant (K FQ) and binding constant (K itc). Conclusion The combination of inhibition kinetics, FQ, DSC and ITC can reasonably characterize the interactions between tea polyphenols and PPA. The galloyl moiety is an important group in catechins and theaflavins in terms of binding with and inhibiting the activity of PPA. PMID:28618113

Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy

PubMed Central

Gepp, Barbara; Lengger, Nina; Bublin, Merima; Hemmer, Wolfgang; Breiteneder, Heimo; Radauer, Christian

2014-01-01

Background Characterization of IgE-binding epitopes of allergens and determination of their patient-specific relevance is crucial for the diagnosis and treatment of allergy. Objective We sought to assess the contribution of specific surface areas of the major birch pollen allergen Bet v 1.0101 to binding IgE of individual patients. Methods Four distinct areas of Bet v 1 representing in total 81% of its surface were grafted onto the scaffold of its homolog, Api g 1.0101, to yield the chimeras Api-Bet-1 to Api-Bet-4. The chimeras were expressed in Escherichia coli and purified. IgE binding of 64 sera from Bet v 1–sensitized subjects with birch pollen allergy was determined by using direct ELISA. Specificity was assessed by means of inhibition ELISA. Results rApi g 1.0101, Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4 bound IgE from 44%, 89%, 80%, 78%, and 48% of the patients, respectively. By comparing the amount of IgE binding to the chimeras and to rApi g 1.0101, 81%, 70%, 75%, and 45% of the patients showed significantly enhanced IgE binding to Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4, respectively. The minority (8%) of the sera revealed enhanced IgE binding exclusively to a single chimera, whereas 31% showed increased IgE binding to all 4 chimeras compared with rApi g 1.0101. The chimeras inhibited up to 70% of IgE binding to rBet v 1.0101, confirming the specific IgE recognition of the grafted regions. Conclusion The Bet v 1–specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is highly patient specific. PMID:24529686

Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy.

PubMed

Gepp, Barbara; Lengger, Nina; Bublin, Merima; Hemmer, Wolfgang; Breiteneder, Heimo; Radauer, Christian

2014-07-01

Characterization of IgE-binding epitopes of allergens and determination of their patient-specific relevance is crucial for the diagnosis and treatment of allergy. We sought to assess the contribution of specific surface areas of the major birch pollen allergen Bet v 1.0101 to binding IgE of individual patients. Four distinct areas of Bet v 1 representing in total 81% of its surface were grafted onto the scaffold of its homolog, Api g 1.0101, to yield the chimeras Api-Bet-1 to Api-Bet-4. The chimeras were expressed in Escherichia coli and purified. IgE binding of 64 sera from Bet v 1-sensitized subjects with birch pollen allergy was determined by using direct ELISA. Specificity was assessed by means of inhibition ELISA. rApi g 1.0101, Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4 bound IgE from 44%, 89%, 80%, 78%, and 48% of the patients, respectively. By comparing the amount of IgE binding to the chimeras and to rApi g 1.0101, 81%, 70%, 75%, and 45% of the patients showed significantly enhanced IgE binding to Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4, respectively. The minority (8%) of the sera revealed enhanced IgE binding exclusively to a single chimera, whereas 31% showed increased IgE binding to all 4 chimeras compared with rApi g 1.0101. The chimeras inhibited up to 70% of IgE binding to rBet v 1.0101, confirming the specific IgE recognition of the grafted regions. The Bet v 1-specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is highly patient specific. Copyright © 2014 The Authors. Published by Mosby, Inc. All rights reserved.

Rosmarinic acid is a novel inhibitor for Hepatitis B virus replication targeting viral epsilon RNA-polymerase interaction.

PubMed

Tsukamoto, Yuta; Ikeda, Sotaro; Uwai, Koji; Taguchi, Riho; Chayama, Kazuaki; Sakaguchi, Takemasa; Narita, Ryo; Yao, Wan-Ling; Takeuchi, Fumihiko; Otakaki, Yukie; Watashi, Koichi; Wakita, Takaji; Kato, Hiroki; Fujita, Takashi

2018-01-01

Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (ε) sequence of HBV pregenomic RNA and viral polymerase (Pol) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of ε-RNA-binding-Pol. Screening showed that 5 out of 3,965 compounds inhibited ε-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited ε-Pol. Structural comparisons between these derivatives implied that the "two phenolic hydroxyl groups at both ends" and the "caffeic acid-like structure" of rosmarinic acid are critical for the inhibition of ε-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting ε-Pol binding.

Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site.

PubMed

Ojeda, Paola; Pérez, Alejandra; Ojeda, Lorena; Vargas-Uribe, Mauricio; Rivas, Coralia I; Salas, Monica; Vera, Juan Carlos; Reyes, Alejandro M

2012-09-01

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.

Inactivation by Phenylglyoxal of the Specific Binding of 1-Naphthyl Acetic Acid with Membrane-Bound Auxin Binding Sites from Maize Coleoptiles

PubMed Central

Navé, Jean-François; Benveniste, Pierre

1984-01-01

The specific binding of 1-[3H]naphthyl acetic acid (NAA) to membrane-bound binding sites from maize (Zea mays cv INRA 258) coleoptiles is inactivated by phenylglyoxal. The inactivation obeys pseudo first-order kinetics. The rate of inactivation is proportional to phenylglyoxal concentration. Under conditions at which significant binding occurs, NAA, R and S-1-naphthyl 2-propionic acids protect the auxin binding site against inactivation by phenylglyoxal. Scatchard analysis shows that the inhibition of binding corresponds to a decrease in the concentration of sites but not in the affinity. The results of the present chemical modification study indicate that at least one arginyl residue is involved in the positively charged recognition site of the carboxylate anion of NAA. PMID:16663499

Autoradiography of P2x ATP receptors in the rat brain.

PubMed Central

Balcar, V. J.; Li, Y.; Killinger, S.; Bennett, M. R.

1995-01-01

1. Binding of a P2x receptor specific radioligand, [3H]-alpha,beta-methylene adenosine triphosphate ([3H]-alpha,beta-MeATP) to sections of rat brain was reversible and association/dissociation parameters indicated that it consisted of two saturable components. Non-specific binding was very low (< 7% at 10 nM ligand concentration). 2. The binding was completely inhibited by suramin (IC50 approximately 14-26 microM) but none of the ligands specific for P2y receptors such as 2-methylthio-adenosine triphosphate (2-methyl-S-ATP) and 2-chloro-adenosine triphosphate (2-C1-ATP) nor 2-methylthio-adenosine diphosphate (2-methyl-S-ADP) a ligand for the P2 receptor on blood platelets ('P2T' type) produced strong inhibitions except for P1,P4-di(adenosine-5')tetraphosphate (Ap4A). 3. Inhibitors of Na+,K(+)-dependent adenosine triphosphatase (ATPase) ouabain, P1-ligand adenosine and an inhibitor of transport of, respectively, adenosine and cyclic nucleotides, dilazep, had no effect. 4. The highest density of P2x binding sites was found to be in the cerebellar cortex but the binding sites were present in all major brain regions, especially in areas known to receive strong excitatory innervation. Images Figure 2 PMID:7670731

Glyceollin I, A Novel Antiestrogenic Phytoalexin Isolated from Activated Soy

USDA-ARS?s Scientific Manuscript database

Glyceollins, a group of novel phytoalexins isolated from activated soy, have recently been demonstrated to be a novel antiestrogen which bind to the estrogen receptor (ER) and inhibit estrogen-induced tumor progression. Our previous publications have focused specifically on inhibition of tumor form...

(+)-3-( sup 123 I)Iodo-MK-801: Synthesis and characterization of binding to the N-methyl-D-aspartate receptor complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ransom, R.W.; Wai-si Eng; Burns, H.D.

1990-01-01

Synthetic methods have been established for preparing high specific activity (+)-3-({sup 123}I)Iodo-MK-801 in high radiochemical yield. The binding of the radiotracer to rat cortical membranes has been examine to assess its potential use as an in vivo imaging agent for the N-methyl-D-aspartate (NMDA) receptor-ion channel complex. Under the conditions of the assay, specific (+)-3-({sup 123}I)Iodo-MK-801 binding to membrane homogenates represented greater than 95% of the total binding. Several structurally distinct, noncompetitive NMDA receptor antagonists inhibited binding with potencies in accordance with their reported inhibitory activity at the receptor complex. The concentration of ({plus minus})-3-Iodo-MK-801 required to inhibit 50% of (+)-3-({supmore » 123}I)Iodo-MK-801 binding (IC{sub 50}) was 3.4 nM when using a low ionic strength assay buffer and 5.5 nM in a physiological buffer. In a thoroughly washed membrane preparation, (+)-3-({sup 123}I)Iodo-MK-801 binding was enhanced by L-glutamate and glycine at concentrations known to activate the NMDA receptor. The results indicate that (+)-3-({sup 123}I)Iodo-MK-801 specifically labels the NMDA receptor complex in rat brain membranes and the retention of high affinity under near physiological assay conditions suggests that it may be useful as a SPECT imaging agent for the receptor in vivo.« less

Substrate specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa.

PubMed

Wylie, J L; Worobec, E A

1993-07-01

Specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa was examined. At a concentration of [14C]glucose near the Vmax of the system, inhibition by maltose, galactose, and xylose was detected. This inhibition is similar to that detected in earlier in vivo studies and correlates with the known specificity of OprB, a glucose-specific porin of P. aeruginosa. At a level of [14C]glucose 100 times lower, only unlabelled glucose inhibited uptake to any extent. This matches the known in vitro specificity of the periplasmic glucose binding protein. These findings were used to explain the discrepancy between earlier in vivo and in vitro results reported in the literature.

Dipetalodipin, a Novel Multifunctional Salivary Lipocalin That Inhibits Platelet Aggregation, Vasoconstriction, and Angiogenesis through Unique Binding Specificity for TXA2, PGF2α, and 15(S)-HETE*

PubMed Central

Assumpção, Teresa C. F.; Alvarenga, Patricia H.; Ribeiro, José M. C.; Andersen, John F.; Francischetti, Ivo M. B.

2010-01-01

Dipetalodipin (DPTL) is an 18 kDa protein cloned from salivary glands of the triatomine Dipetalogaster maxima. DPTL belongs to the lipocalin superfamily and has strong sequence similarity to pallidipin, a salivary inhibitor of collagen-induced platelet aggregation. DPTL expressed in Escherichia coli was found to inhibit platelet aggregation by collagen, U-46619, or arachidonic acid without affecting aggregation induced by ADP, convulxin, PMA, and ristocetin. An assay based on incubation of DPTL with small molecules (e.g. prostanoids, leukotrienes, lipids, biogenic amines) followed by chromatography, mass spectrometry, and isothermal titration calorimetry showed that DPTL binds with high affinity to carbocyclic TXA2, TXA2 mimetic (U-46619), TXB2, PGH2 mimetic (U-51605), PGD2, PGJ2, and PGF2α. It also interacts with 15(S)-HETE, being the first lipocalin described to date to bind to a derivative of 15-lipoxygenase. Binding was not observed to other prostaglandins (e.g. PGE1, PGE2, 8-iso-PGF2α, prostacyclin), leukotrienes (e.g,. LTB4, LTC4, LTD4, LTE4), HETEs (e.g. 5(S)-HETE, 12(S)-HETE, 20-HETE), lipids (e.g. arachidonic acid, PAF), and biogenic amines (e.g. ADP, serotonin, epinephrine, norepinephrine, histamine). Consistent with its binding specificity, DPTL prevents contraction of rat uterus stimulated by PGF2α and induces relaxation of aorta previously contracted with U-46619. Moreover, it inhibits angiogenesis mediated by 15(S)-HETE and did not enhance inhibition of collagen-induced platelet aggregation by SQ29548 (TXA2 antagonist) and indomethacin. A 3-D model for DPTL and pallidipin is presented that indicates the presence of a conserved Arg39 and Gln135 in the binding pocket of both lipocalins. Results suggest that DPTL blocks platelet aggregation, vasoconstriction, and angiogenesis through binding to distinct eicosanoids involved in inflammation. PMID:20889972

Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sun Young; Song, Kyung-A; Samsung Biomedical Research Institute

Highlights: Black-Right-Pointing-Pointer Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. Black-Right-Pointing-Pointer A small molecule and a peptide as EBNA1 dimerization inhibitors identified. Black-Right-Pointing-Pointer Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. Black-Right-Pointing-Pointer Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation.more » In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-J{kappa} binding to the J{kappa} site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with EBNA1 in vitro, and repressed EBNA1-dependent transcription in vivo. Collectively, this study describes two novel inhibitors of EBNA1 dimerization. This study demonstrates that EBNA1 homodimerization can be effectively targeted by a small molecule or peptide.« less

Metalloregulatory Proteins: Metal Selectivity and Allosteric Switching

PubMed Central

Caballero, Hermes Reyes; Campanello, Gregory C.; Giedroc, David P.

2011-01-01

Prokaryotic organisms have evolved an impressive capacity to quickly adapt to a changing and challenging microenvironment in which the availability of both biologically required and non-essential transition metal ions can vary dramatically. In all bacteria, a panel of metalloregulatory proteins control the expression of genes encoding membrane transporters and metal trafficking proteins, that collectively manage metal homeostasis and resistance. These “metal sensors” are specialized allosteric proteins, in which the direct binding of a specific or small number of “cognate” metal ion(s) drives a conformational change in the regulator that allosterically activates or inhibits operator DNA binding, or alternatively, distorts the promoter structure thereby converting a poor promoter to a strong one. In this review, we discuss our current understanding of the features that control metal specificity of the allosteric response in these systems, and the role that structure, thermodynamics and conformational dynamics play in mediating allosteric activation or inhibition of DNA binding. PMID:21511390

Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.

PubMed

Birchfield, N B; Casida, J E

1996-01-01

Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

DOE PAGES

Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; ...

2016-03-14

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface.more » Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.« less

Isolation and characterization at cholinergic nicotinic receptors of a neurotoxin from the venom of the Acanthophis sp. Seram death adder.

PubMed

Wickramaratna, Janith C; Fry, Bryan G; Loiacono, Richard E; Aguilar, Marie-Isabel; Alewood, Paul F; Hodgson, Wayne C

2004-07-15

The present study describes the isolation of the first neurotoxin (acantoxin IVa) from Acanthophis sp. Seram death adder venom and an examination of its activity at nicotinic acetylcholine receptor (nAChR) subtypes. Acantoxin IVa (MW 6815; 0.1-1.0 microM) caused concentration-dependent inhibition of indirect twitches (0.1 Hz, 0.2 ms, supramaximal V) and inhibited contractile responses to exogenous nicotinic agonists in the chick biventer cervicis nerve-muscle, confirming that this toxin is a postsynaptic neurotoxin. Acantoxin IVa (1-10 nM) caused pseudo-irreversible antagonism at skeletal muscle nAChR with an estimated pA2 of 8.36+/-0.17. Acantoxin IVa was approximately two-fold less potent than the long-chain (Type II) neurotoxin, alpha-bungarotoxin. With a pKi value of 4.48, acantoxin IVa was approximately 25,000 times less potent than alpha-bungarotoxin at alpha7-type neuronal nAChR. However, in contrast to alpha-bungarotoxin, acantoxin IVa completely inhibited specific [3H]-methyllycaconitine (MLA) binding in rat hippocampus homogenate. Acantoxin IVa had no activity at ganglionic nAChR, alpha4beta2 subtype neuronal nAChR or cytisine-resistant [3H]-epibatidine binding sites. While long-chain neurotoxin resistant [3H]-MLA binding in hippocampus homogenate requires further investigation, we have shown that a short-chain (Type I) neurotoxin is capable of fully inhibiting specific [3H]-MLA binding.

Heparin (GAG-hed) inhibits LCR activity of human papillomavirus type 18 by decreasing AP1 binding.

PubMed

Villanueva, Rita; Morales-Peza, Néstor; Castelán-Sánchez, Irma; García-Villa, Enrique; Tapia, Rocio; Cid-Arregui, Angel; García-Carrancá, Alejandro; López-Bayghen, Esther; Gariglio, Patricio

2006-08-31

High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data suggest that GAG-hed could have antitumoral and antiviral activity mainly by inhibiting AP1 binding to the HPV18-LCR.

Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

PubMed

Philips, Brian J; Ansell, Pete J; Newton, Leslie G; Harada, Nobuhiro; Honda, Shin-Ichiro; Ganjam, Venkataseshu K; Rottinghaus, George E; Welshons, Wade V; Lubahn, Dennis B

2004-06-01

Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

Molecular mechanism of pancreatic tumor metastasis inhibition by Gd@C 82(OH) 22 and its implication for de novo design of nanomedicine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, S. -g.; Zhou, G.; Yang, P.

2012-09-18

Pancreatic adenocarcinoma is the most lethal of the solid tumors and the fourth-leading cause of cancer-related death in North America. Matrix metalloproteinases (MMPs) have long been targeted as a potential anticancer therapy because of their seminal role in angiogenesis and extracellular matrix (ECM) degradation of tumor survival and invasion. However, the inhibition specificity to MMPs and the molecular-level understanding of the inhibition mechanism remain largely unresolved. Here, we found that endohedral metallofullerenol Gd@C 82(OH) 22 can successfully inhibit the neoplastic activity with experiments at animal, tissue, and cellular levels. Gd@C 82(OH) 22 effectively blocks tumor growth in human pancreatic cancermore » xenografts in a nude mouse model. Enzyme activity assays also show Gd@C 82(OH) 22 not only suppresses the expression of MMPs but also significantly reduces their activities. We then applied large-scale molecular-dynamics simulations to illustrate the molecular mechanism by studying the Gd@C 82(OH) 22–MMP-9 interactions in atomic detail. Our data demonstrated that Gd@C 82(OH) 22 inhibits MMP-9 mainly via an exocite interaction, whereas the well-known zinc catalytic site only plays a minimal role. Steered by nonspecific electrostatic, hydrophobic, and specific hydrogen-bonding interactions, Gd@C 82(OH) 22 exhibits specific binding modes near the ligand-specificity loop S1', thereby inhibiting MMP-9 activity. Both the suppression of MMP expression and specific binding mode make Gd@C 82(OH) 22 a potentially more effective nanomedicine for pancreatic cancer than traditional medicines, which usually target the proteolytic sites directly but fail in selective inhibition. Finally, our findings provide insights for de novo design of nanomedicines for fatal diseases such as pancreatic cancer.« less

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

PubMed

Hamm, Jorg; Alessi, Dario R; Biondi, Ricardo M

2002-11-29

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

Human H3N2 Influenza Viruses Isolated from 1968 To 2012 Show Varying Preference for Receptor Substructures with No Apparent Consequences for Disease or Spread

PubMed Central

Gulati, Shelly; Smith, David F.; Cummings, Richard D.; Couch, Robert B.; Griesemer, Sara B.; St. George, Kirsten; Webster, Robert G.; Air, Gillian M.

2013-01-01

It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2–6 to the next sugar, that avian influenza viruses bind glycans containing the α2–3 linkage, and that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2–6(Galβ1–4GlcNAc)3 in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2–3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2–3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population. PMID:23805213

Competitive Inhibition Mechanism of Acetylcholinesterase without Catalytic Active Site Interaction: Study on Functionalized C60 Nanoparticles via in Vitro and in Silico Assays.

PubMed

Liu, Yanyan; Yan, Bing; Winkler, David A; Fu, Jianjie; Zhang, Aiqian

2017-06-07

Acetylcholinesterase (AChE) activity regulation by chemical agents or, potentially, nanomaterials is important for both toxicology and pharmacology. Competitive inhibition via direct catalytic active sites (CAS) binding or noncompetitive inhibition through interference with substrate and product entering and exiting has been recognized previously as an AChE-inhibition mechanism for bespoke nanomaterials. The competitive inhibition by peripheral anionic site (PAS) interaction without CAS binding remains unexplored. Here, we proposed and verified the occurrence of a presumed competitive inhibition of AChE without CAS binding for hydrophobically functionalized C 60 nanoparticles (NPs) by employing both experimental and computational methods. The kinetic inhibition analysis distinguished six competitive inhibitors, probably targeting the PAS, from the pristine and hydrophilically modified C 60 NPs. A simple quantitative nanostructure-activity relationship (QNAR) model relating the pocket accessible length of substituent to inhibition capacity was then established to reveal how the geometry of the surface group decides the NP difference in AChE inhibition. Molecular docking identified the PAS as the potential binding site interacting with the NPs via a T-shaped plug-in mode. Specifically, the fullerene core covered the enzyme gorge as a lid through π-π stacking with Tyr72 and Trp286 in the PAS, while the hydrophobic ligands on the fullerene surface inserted into the AChE active site to provide further stability for the complexes. The modeling predicted that inhibition would be severely compromised by Tyr72 and Trp286 deletions, and the subsequent site-directed mutagenesis experiments proved this prediction. Our results demonstrate AChE competitive inhibition of NPs without CAS participation to gain further understanding of both the neurotoxicity and the curative effect of NPs.

p65 fragments, homologous to the C2 region of protein kinase C, bind to the intracellular receptors for protein kinase C.

PubMed

Mochly-Rosen, D; Miller, K G; Scheller, R H; Khaner, H; Lopez, J; Smith, B L

1992-09-08

Receptors for activated protein kinase C (RACKs) have been isolated from the particulate cell fraction of heart and brain. We previously demonstrated that binding of protein kinase C (PKC) to RACKs requires PKC activators and is via a site on PKC that is distinct from the substrate binding site. Here, we examine the possibility that the C2 region in the regulatory domain of PKC is involved in binding of PKC to RACKs. The synaptic vesicle-specific p65 protein contains two regions homologous to the C2 region of PKC. We found that three p65 fragments, containing either one or two of these PKC C2 homologous regions, bound to highly purified RACKs. Binding of the p65 fragments and PKC to RACKs was mutually exclusive; preincubation of RACKs with the p65 fragments inhibited PKC binding, and preincubation of RACKs with PKC inhibited binding of the p65 fragments. Preincubation of the p65 fragments with a peptide resembling the PKC binding site on RACKs also inhibited p65 binding to RACKs, suggesting that PKC and p65 bind to the same or nearby regions on RACKs. Since the only homologous region between PKC and the p65 fragments is the C2 region, these results suggest that the C2 region on PKC contains at least part of the RACK binding site.

Selective chemical binding enhances cesium tolerance in plants through inhibition of cesium uptake

PubMed Central

Adams, Eri; Chaban, Vitaly; Khandelia, Himanshu; Shin, Ryoung

2015-01-01

High concentrations of cesium (Cs+) inhibit plant growth but the detailed mechanisms of Cs+ uptake, transport and response in plants are not well known. In order to identify small molecules with a capacity to enhance plant tolerance to Cs+, chemical library screening was performed using Arabidopsis. Of 10,000 chemicals tested, five compounds were confirmed as Cs+ tolerance enhancers. Further investigation and quantum mechanical modelling revealed that one of these compounds reduced Cs+ concentrations in plants and that the imidazole moiety of this compound bound specifically to Cs+. Analysis of the analogous compounds indicated that the structure of the identified compound is important for the effect to be conferred. Taken together, Cs+ tolerance enhancer isolated here renders plants tolerant to Cs+ by inhibiting Cs+ entry into roots via specific binding to the ion thus, for instance, providing a basis for phytostabilisation of radiocesium-contaminated farmland. PMID:25740624

Selective chemical binding enhances cesium tolerance in plants through inhibition of cesium uptake.

PubMed

Adams, Eri; Chaban, Vitaly; Khandelia, Himanshu; Shin, Ryoung

2015-03-05

High concentrations of cesium (Cs(+)) inhibit plant growth but the detailed mechanisms of Cs(+) uptake, transport and response in plants are not well known. In order to identify small molecules with a capacity to enhance plant tolerance to Cs(+), chemical library screening was performed using Arabidopsis. Of 10,000 chemicals tested, five compounds were confirmed as Cs(+) tolerance enhancers. Further investigation and quantum mechanical modelling revealed that one of these compounds reduced Cs(+) concentrations in plants and that the imidazole moiety of this compound bound specifically to Cs(+). Analysis of the analogous compounds indicated that the structure of the identified compound is important for the effect to be conferred. Taken together, Cs(+) tolerance enhancer isolated here renders plants tolerant to Cs(+) by inhibiting Cs(+) entry into roots via specific binding to the ion thus, for instance, providing a basis for phytostabilisation of radiocesium-contaminated farmland.

Phosphorylation of NHE3-S719 regulates NHE3 activity through the formation of multiple signaling complexes

PubMed Central

Sarker, Rafiquel; Cha, Boyoung; Kovbasnjuk, Olga; Cole, Robert; Gabelli, Sandra; Tse, Chung Ming; Donowitz, Mark

2017-01-01

Casein kinase 2 (CK2) binds to the NHE3 C-terminus and constitutively phosphorylates a downstream site (S719) that accounts for 40% of basal NHE3 activity. The role of CK2 in regulation of NHE3 activity in polarized Caco-2/bbe cells was further examined by mutation of NHE3-S719 to A (not phosphorylated) or D (phosphomimetic). NHE3-S719A but not -S719D had multiple changes in NHE3 activity: 1) reduced basal NHE3 activity—specifically, inhibition of the PI3K/AKT-dependent component; 2) reduced acute stimulation of NHE3 activity by LPA/LPA5R stimulation; and 3) reduced acute inhibition of NHE3 activity—specifically, elevated Ca2+ related (carbachol/Ca2+ ionophore), but there was normal inhibition by forskolin and hyperosmolarity. The S719A mutant had reduced NHE3 complex size, reduced expression in lipid rafts, increased BB mobile fraction, and reduced binding to multiple proteins that bind throughout the NHE3 intracellular C-terminus, including calcineurin homologous protein, the NHERF family and SNX27 (related PDZ domains). These studies show that phosphorylation of the NHE3 at a single amino acid in the distal part of the C-terminus affects multiple aspects of NHE3 complex formation and changes the NHE3 lipid raft distribution, which cause changes in specific aspects of basal as well as acutely stimulated and inhibited Na+/H+ exchange activity. PMID:28495796

A Novel High-Mannose Specific Lectin from the Green Alga Halimeda renschii Exhibits a Potent Anti-Influenza Virus Activity through High-Affinity Binding to the Viral Hemagglutinin

PubMed Central

Mu, Jinmin; Hirayama, Makoto; Sato, Yuichiro; Morimoto, Kinjiro; Hori, Kanji

2017-01-01

We have isolated a novel lectin, named HRL40 from the green alga Halimeda renschii. In hemagglutination-inhibition test and oligosaccharide-binding experiment with 29 pyridylaminated oligosaccharides, HRL40 exhibited a strict binding specificity for high-mannose N-glycans having an exposed (α1-3) mannose residue in the D2 arm of branched mannosides, and did not have an affinity for monosaccharides and other oligosaccharides examined, including complex N-glycans, an N-glycan core pentasaccharide, and oligosaccharides from glycolipids. The carbohydrate binding profile of HRL40 resembled those of Type I high-mannose specific antiviral algal lectins, or the Oscillatoria agardhii agglutinin (OAA) family, which were previously isolated from red algae and a blue-green alga (cyanobacterium). HRL40 potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells with half-maximal effective dose (ED50) of 2.45 nM through high-affinity binding to a viral envelope hemagglutinin (KD, 3.69 × 10−11 M). HRL40 consisted of two isolectins (HRL40-1 and HRL40-2), which could be separated by reverse-phase HPLC. Both isolectins had the same molecular weight of 46,564 Da and were a disulfide -linked tetrameric protein of a 11,641 Da polypeptide containing at least 13 half-cystines. Thus, HRL40, which is the first Type I high-mannose specific antiviral lectin from the green alga, had the same carbohydrate binding specificity as the OAA family, but a molecular structure distinct from the family. PMID:28813016

The inhibition of mitochondrial calcium transport by lanthanides and Ruthenium Red

PubMed Central

Reed, Ken C.; Bygrave, Fyfe L.

1974-01-01

An EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-quench technique was developed for measuring initial rates of 45Ca2+ transport by rat liver mitochondria. This method was used in conjunction with studies of Ca2+-stimulated respiration to examine the mechanisms of inhibition of Ca2+ transport by the lanthanides and Ruthenium Red. Ruthenium Red inhibits Ca2+ transport non-competitively with Ki 3×10−8m; there are 0.08nmol of carrier-specific binding sites/mg of protein. The inhibition by La3+ is competitive (Ki=2×10−8m); the concentration of lanthanide-sensitive sites is less than 0.001nmol/mg of protein. A further difference between their modes of action is that lanthanide inhibition diminishes with time whereas that by Ruthenium Red does not. Binding studies showed that both classes of inhibitor bind to a relatively large number of external sites (probably identical with the `low-affinity' Ca2+-binding sites). La3+ competes with Ruthenium Red for most of these sites, but a small fraction of the bound Ruthenium Red (less than 2nmol/mg of protein) is not displaced by La3+. The results are discussed briefly in relation to possible models for a Ca2+ carrier. PMID:4375957

Follicle-stimulating hormone (FSH) unmasks specific high affinity FSH-binding sites in cell-free membrane preparations of porcine granulosa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ford, K.A.; LaBarbera, A.R.

1988-11-01

The purpose of these studies was to determine whether changes in FSH receptors correlated with FSH-induced attenuation of FSH-responsive adenylyl cyclase in immature porcine granulosa cells. Cells were incubated with FSH (1-1000 ng/ml) for up to 24 h, treated with acidified medium (pH 3.5) to remove FSH bound to cells, and incubated with (125I)iodo-porcine FSH to quantify FSH-binding sites. FSH increased binding of FSH in a time-, temperature-, and FSH concentration-dependent manner. FSH (200 ng/ml) increased binding approximately 4-fold within 16 h. Analysis of equilibrium saturation binding data indicated that the increase in binding sites reflected a 2.3-fold increase inmore » receptor number and a 5.4-fold increase in apparent affinity. The increase in binding did not appear to be due to 1) a decrease in receptor turnover, since the basal rate of turnover appeared to be very slow; 2) an increase in receptor synthesis, since agents that inhibit protein synthesis and glycosylation did not block the increase in binding; or 3) an increase in intracellular receptors, since agents that inhibit cytoskeletal components had no effect. Agents that increase intracellular cAMP did not affect FSH binding. The increase in binding appeared to result from unmasking of cryptic FSH-binding sites, since FSH increased binding in cell-free membrane preparations to the same extent as in cells. Unmasking of cryptic sites was hormone specific, and the sites bound FSH specifically. Unmasking of sites was reversible in a time- and temperature-dependent manner after removal of bound FSH. The similarity between the FSH dose-response relationships for unmasking of FSH-binding sites and attenuation of FSH-responsive cAMP production suggests that the two processes are functionally linked.« less

Agmatine is transported into liver mitochondria by a specific electrophoretic mechanism

PubMed Central

Salvi, Mauro; Battaglia, Valentina; Mancon, Mario; Colombatto, Sebastiano; Cravanzola, Carlo; Calheiros, Rita; Marques, Maria P. M.; Grillo, Maria A.; Toninello, Antonio

2006-01-01

Agmatine, a divalent diamine with two positive charges at physiological pH, is transported into the matrix of liver mitochondria by an energy-dependent mechanism the driving force of which is ΔΨ (electrical membrane potential). Although this process showed strict electrophoretic behaviour, qualitatively similar to that of polyamines, agmatine is most probably transported by a specific uniporter. Shared transport with polyamines by means of their transporter is excluded, as divalent putrescine and cadaverine are ineffective in inhibiting agmatine uptake. Indeed, the use of the electroneutral transporter of basic amino acids can also be discarded as ornithine, arginine and lysine are completely ineffective at inducing the inhibition of agmatine uptake. The involvement of the monoamine transporter or the existence of a leak pathway are also unlikely. Flux-voltage analysis and the determination of activation enthalpy, which is dependent upon the valence of agmatine, are consistent with the hypothesis that the mitochondrial agmatine transporter is a channel or a single-binding centre-gated pore. The transport of agmatine was non-competitively inhibited by propargylamines, in particular clorgilyne, that are known to be inhibitors of MAO (monoamine oxidase). However, agmatine is normally transported in mitoplasts, thus excluding the involvement of MAO in this process. The I2 imidazoline receptor, which binds agmatine to the mitochondrial membrane, can also be excluded as a possible transporter since its inhibitor, idazoxan, was ineffective at inducing the inhibition of agmatine uptake. Scatchard analysis of membrane binding revealed two types of binding site, S1 and S2, both with mono-co-ordination, and exhibiting high-capacity and low-affinity binding for agmatine compared with polyamines. Agmatine transport in liver mitochondria may be of physiological importance as an indirect regulatory system of cytochrome c oxidase activity and as an inducer mechanism of mitochondrial-mediated apoptosis. PMID:16509824

Specific strychnine binding sites on acrosome-associated membranes of golden hamster spermatozoa.

PubMed

Llanos, Miguel N; Ronco, Ana M; Aguirre, María C

2003-06-27

This study demonstrates for the first time, that membrane vesicles originated from the hamster sperm head after the occurrence of the acrosome reaction, possess specific strychnine binding sites. [3H]Strychnine binding was saturable and reversible, being displaced by unlabeled strychnine (IC(50)=26.7+/-2.3 microM). Kinetic analysis revealed one binding site with K(d)=120nM and B(max)=142fmol/10(6) spermatozoa. Glycine receptor agonists beta-alanine and taurine inhibited strychnine binding by 20-30%. Surprisingly, glycine stimulated binding by about 40-50%. Results obtained in this study strongly suggest the presence of glycine receptors-with distinctive kinetic properties on the periacrosomal plasma membrane of hamster spermatozoa. Localization of this receptor fits well with its previously proposed role in acrosomal exocytosis during mammalian fertilization.

The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin

PubMed Central

Santos, Kira L.; Vento, Megan A; Wright, John W.; Speth, Robert C.

2013-01-01

A novel, non-AT1, non-AT2 brain binding site for angiotensin peptides that is unmasked by p-chloromercuribenzoate (PCMB) has been identified as a membrane associated variant of neurolysin. The ability of different organic and inorganic oxidative and sulfhydryl reactive agents to unmask or inhibit 125I-Sar1Ile8 angiotensin II (SI-Ang II) binding to this site was presently examined. In tissue membranes from homogenates of rat brain and testis incubated in assay buffer containing losartan (10 μM) and PD123319 (10 μM) plus 100 μM PCMB, 5 of the 39 compounds tested inhibited 125I-SI Ang II binding in brain and testis. Mersalyl acid, mercuric chloride (HgCl2) and silver nitrate (AgNO3) most potently inhibited 125I-SI Ang II binding with IC50’s ~1–20 μM This HgCl2 inhibition was independent of any interaction of HgCl2 with angiotensin II (Ang II) based on the lack of effect of HgCl2 on the dipsogenic effects of intracerebroventricularly administered Ang II and 125I-SI Ang II binding to AT1 receptors in the liver. Among sulfhydryl reagents, cysteamine and reduced glutathione (GSH), but not oxidized glutathione (GSSG) up to 1 mM, inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis. Thimerosal and 4-hydroxymercuribenzoate moderately inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis at 100 μM; however, they also unmasked non-AT1, non-AT2 binding independent of PCMB. 4-hydroxybenzoic acid did not promote 125 I-SI Ang II binding to this binding site indicating that only specific organomercurial compounds can unmask the binding site. The common denominator for all of these interacting substances is the ability to bind to protein cysteine sulfur. Comparison of cysteines between neurolysin and the closely related enzyme thimet oligopeptidase revealed an unconserved cysteine (cys650, based on the full length variant) in the proposed ligand binding channel (Brown et al., 2001) [1] near the active site of neurolysin. It is proposed that the mercuric ion in PCMB and closely related organomercurial compounds binds to cys650, while the acidic anion forms an ionic bond with a nearby arginine or lysine along the channel to effect a conformational change in neurolysin that promotes Ang II binding. PMID:23511333

Mercury(II) binds to both of chymotrypsin's histidines, causing inhibition followed by irreversible denaturation/aggregation.

PubMed

Stratton, Amanda; Ericksen, Matthew; Harris, Travis V; Symmonds, Nick; Silverstein, Todd P

2017-02-01

The toxicity of mercury is often attributed to its tight binding to cysteine thiolate anions in vital enzymes. To test our hypothesis that Hg(II) binding to histidine could be a significant factor in mercury's toxic effects, we studied the enzyme chymotrypsin, which lacks free cysteine thiols; we found that chymotrypsin is not only inhibited, but also denatured by Hg(II). We followed the aggregation of denatured enzyme by the increase in visible absorbance due to light scattering. Hg(II)-induced chymotrypsin precipitation increased dramatically above pH 6.5, and free imidazole inhibited this precipitation, implicating histidine-Hg(II) binding in the process of chymotrypsin denaturation/aggregation. Diethylpyrocarbonate (DEPC) blocked chymotrypsin's two histidines (his 40 and his 57 ) quickly and completely, with an IC 50 of 35 ± 6 µM. DEPC at 350 µM reduced the hydrolytic activity of chymotrypsin by 90%, suggesting that low concentrations of DEPC react with his 57 at the active site catalytic triad; furthermore, DEPC below 400 µM enhanced the Hg(II)-induced precipitation of chymotrypsin. We conclude that his 57 reacts readily with DEPC, causing enzyme inhibition and enhancement of Hg(II)-induced aggregation. Above 500 µM, DEPC inhibited Hg(II)-induced precipitation, and [DEPC] >2.5 mM completely protected chymotrypsin against precipitation. This suggests that his 40 reacts less readily with DEPC, and that chymotrypsin denaturation is caused by Hg(II) binding specifically to the his 40 residue. Finally, we show that Hg(II)-histidine binding may trigger hemoglobin aggregation as well. Because of results with these two enzymes, we suggest that metal-histidine binding may be key to understanding all heavy metal-induced protein aggregation. © 2017 The Protein Society.

Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA.

PubMed

Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

2015-07-13

We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. © Crown copyright 2015.

Binding of phycoerythrin and its conjugates to murine low affinity receptors for immunoglobulin G.

PubMed

Takizawa, F; Kinet, J P; Adamczewski, M

1993-06-18

Conjugates of R-phycoerythrin are widely used for immunohistochemistry, especially for two-color flow cytometry. Their use is however limited by their apparent tendency to bind non-specifically. Using cells transfected with cDNAs for the murine low affinity receptors for immunoglobulin G (Fc gamma RII and -III) and cells naturally expressing these receptors, we demonstrate that R-phycoerythrin and its conjugates bind specifically and inhibitably to Fc gamma RII and -III. Immunofluorescence stainings of cells bearing these receptors, such as macrophages, monocytes, neutrophils, mast cells, subsets of T cells, and natural killer cells, may therefore not reflect the binding of antibody to antigen, but rather the binding of R-phycoerythrin to the receptors.

Identification of a New Functional Domain in Angiopoietin-like 3 (ANGPTL3) and Angiopoietin-like 4 (ANGPTL4) Involved in Binding and Inhibition of Lipoprotein Lipase (LPL)S⃞

PubMed Central

Lee, E-Chiang; Desai, Urvi; Gololobov, Gennady; Hong, Seokjoo; Feng, Xiao; Yu, Xuan-Chuan; Gay, Jason; Wilganowski, Nat; Gao, Cuihua; Du, Ling-Ling; Chen, Joan; Hu, Yi; Zhao, Sharon; Kirkpatrick, Laura; Schneider, Matthias; Zambrowicz, Brian P.; Landes, Greg; Powell, David R.; Sonnenburg, William K.

2009-01-01

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln29–His53, which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu32–His55. We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3-/- mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia. PMID:19318355

Characterizing the Anti-HIV Activity of Papuamide A

PubMed Central

Andjelic, Cynthia D; Planelles, Vicente; Barrows, Louis R

2008-01-01

Papuamide A is representative of a class of marine derived cyclic depsipeptides, reported to have cytoprotective activity against HIV-1 in vitro. We show here that papuamide A acts as an entry inhibitor, preventing human immunodeficiency virus infection of host cells and that this inhibition is not specific to R5 or X4 tropic virus. This inhibition of viral entry was determined to not be due to papuamide A binding to CD4 or HIV gp120, the two proteins involved in the cell-virus recognition and binding. Furthermore, papuamide A was able to inhibit HIV pseudotype viruses expressing envelope glycoproteins from vesicular stomatitis virus or amphotropic murine leukemia virus indicating the mechanism of viral entry inhibition is not HIV-1 envelope glycoprotein specific. Time delayed addition studies with the pseudotyped viruses show that papuamide A inhibits viral infection only at the initial stage of the viral life cycle. Additionally, pretreatment studies revealed that the virus, and not the cell, is the target of papuamide A’s action. Together, these results suggest a direct virucidal mechanism of HIV-1 inhibition by papuamide A. We also demonstrate here that the other papuamides (B-D) are able to inhibit viral entry indicating that the free amino moiety of 2,3-diaminobutanoic acid residue is not required for the virucidal activity. PMID:19172193

Inhibition of vincristine binding to plasma membrane vesicles from daunorubicin-resistant Ehrlich ascites cells by multidrug resistance modulators.

PubMed Central

Sehested, M.; Jensen, P. B.; Skovsgaard, T.; Bindslev, N.; Demant, E. J.; Friche, E.; Vindeløv, L.

1989-01-01

The multidrug resistance (MDR) phenotype is presumed to be mostly dependent on changes in the resistant cell plasma membrane, notably the emergence of a 170 kDa glycoprotein called P-glycoprotein, which facilitate increased drug efflux. We have previously demonstrated that ATP-enhanced binding of vincristine (VCR) to plasma membrane vesicles is much greater in MDR than in wild type cells. The present study has shown that VCR binding to MDR Ehrlich ascites tumour cell plasma membrane vesicles is inhibited 50% most efficiently by quinidine (0.5 microM) followed by verapamil (4.1 microM) and trifluoperazine (23.2 microM). This is the reverse order of the effect on whole cells where a ranking of efficiency in terms of enhancement of VCR accumulation, inhibition of VCR efflux, DNA perturbation and modulation of resistance in a clonogenic assay, was trifluoperazine greater than or equal to verapamil much greater than quinidine. The detergent Tween 80 inhibited VCR binding to plasma membrane vesicles at 0.001% v/v which agreed with the level which modulated resistance and increased VCR accumulation in whole cells. No effect was observed on daunorubicin binding to MDR plasma membrane vesicles after incubation with either Tween 80 (up to 0.1% v/v) or verapamil (up to 25 microM). We conclude that the effect of a modulating drug in reversing resistance to VCR correlates with its ability to raise intracellular VCR levels but not with its capability to inhibit VCR binding to the plasma membrane. Thus, enhancement of VCR accumulation in MDR cells is hardly solely due to competition for a drug binding site on P-glycoprotein. Furthermore, the lack of a demonstrable effect on daunorubicin binding to the plasma membrane by modulators points to transport mechanisms which do not utilise specific drug binding to the plasma membrane. PMID:2605092

The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Humphreys, C.J.

1989-01-01

The plasmalemmal serotonin transporter uses transmembrane gradients of Na{sup +}, Cl{sup {minus}} and K{sup +} to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na{sup +}. Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na{sup +} and Cl{sup {minus}}, the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized,more » purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na{sup +} binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl{sup {minus}}. Cl{sup {minus}} enhances the transporters affinity for imipramine, as well as for Na{sup +}. At concentrations in the range of its K{sub M} for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na{sup +}-independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both ({sup 3}H)imipramine binding and ({sup 3}H)serotonin transport.« less

CCL22-specific Antibodies Reveal That Engagement of Two Distinct Binding Domains on CCL22 Is Required for CCR4-mediated Function.

PubMed

Santulli-Marotto, Sandra; Wheeler, John; Lacy, Eilyn R; Boakye, Ken; Luongo, Jennifer; Wu, Sheng-Jiun; Ryan, Mary

2015-12-01

CCL22 inactivation in vivo occurs by cleavage at the N-terminus; however, it is unclear whether this encompasses the entire site of CCR4 interaction. CCL17 also binds CCR4 and its function requires binding via two discrete binding sites. Using monoclonal antibodies (MAbs), we report that there are two separate sites on CCL22 that are required for CCR4-mediated function. The CCL22-specific antibodies bind with affinities of 632 ± 297 pM (MC2B7) and 308 ± 43 pM (MAB4391) and neither exhibited detectable binding to CCL17. Both antibodies are comparable in their ability to inhibit CCL22-mediated calcium mobilization; however, competition binding studies demonstrate that MC2B7 and MAB4391 bind to distinct epitopes on CCL22. Both antibodies inhibit function through CCR4, which is demonstrated by loss of β-arrestin recruitment in a reporter cell line. In both assays, blocking either site independently abolished CCL22 function, suggesting that concurrent engagement of both sites with CCR4 is necessary for function. This is the first demonstration that CCL22 has two distinct binding sites that are required for CCR4 function. These antibodies are valuable tools for better understanding the interaction and function of CCL22 and CCR4 and will potentially help further understanding of the differential outcomes of CCL17 and CCL22 interaction with CCR4.

Fragment-based screen against HIV protease.

PubMed

Perryman, Alexander L; Zhang, Qing; Soutter, Holly H; Rosenfeld, Robin; McRee, Duncan E; Olson, Arthur J; Elder, John E; Stout, C David

2010-03-01

We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 A resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the 'exo site' adjacent to the Gly(16)Gly(17)Gln(18)loop where the amide of Gly(17)is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys(14)and Leu(63). Another fragment, indole-6-carboxylic acid, binds on the 'outside/top of the flap' via hydrophobic contacts with Trp(42), Pro(44), Met(46), and Lys(55), a hydrogen bond with Val(56), and a salt-bridge with Arg(57). 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target.

Fragment-Based Screen against HIV Protease

PubMed Central

Perryman, A. L.; Zhang, Q.; Soutter, H. H.; Rosenfeld, R.; McRee, D. E.; Olson, A. J.; Elder, J. E.; Stout, C. D.

2009-01-01

We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 Å resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the ‘exo site’ adjacent to the Gly16Gly17Gln18 loop where the amide of Gly17 is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys14 and Leu63. Another fragment, indole-6-carboxylic acid, binds on the ‘outside/top of the flap’ via hydrophobic contacts with Trp42, Pro44, Met46, and Lys55, a hydrogen bond with Val56, and a salt-bridge with Arg57. 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target. PMID:20659109

Inhibiting platelets aggregation could aggravate the acute infection caused by Staphylococcus aureus.

PubMed

Zhang, Xin; Liu, Yu; Gao, Yaping; Dong, Jie; Mu, Chunhua; Lu, Qiang; Shao, Ningsheng; Yang, Guang

2011-01-01

Several fibrinogen binding proteins (Fibs) play important roles in the pathogenesis of Staphylococcus aureus (S. aureus). Most Fibs can promote the aggregation of platelets during infection, but the extracellular fibrinogen-binding protein (Efb) is an exception. It is reported that Efb can specifically bind fibrinogen and inhibit the aggregation of platelet with its N terminal. However, the biological significance of platelet aggregation inhibition in the infection caused by S. aureus is unclear until now. Here, we demonstrated that the persistence and aggregation of platelets were important for killing S. aureus in whole blood. It was found that the N terminal of Efb (EfbN) and platelets inhibitors could increase the survival of S. aureus in whole blood. The study in vivo also showed that EfbN and platelets inhibitors could reduce the killing of S. aureus and increase the lethality rate of S. aureus in the acute infection mouse model.

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

PubMed

Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

1990-09-01

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

Highly selective inhibition of myosin motors provides the basis of potential therapeutic application

PubMed Central

Sirigu, Serena; Hartman, James J.; Ropars, Virginie; Clancy, Sheila; Wang, Xi; Chuang, Grace; Qian, Xiangping; Lu, Pu-Ping; Barrett, Edward; Rudolph, Karin; Royer, Christopher; Morgan, Bradley P.; Stura, Enrico A.; Malik, Fady I.; Houdusse, Anne M.

2016-01-01

Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the “recovery stroke” transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents. PMID:27815532

From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

PubMed Central

Cígler, Petr; Kožíšek, Milan; Řezáčová, Pavlína; Brynda, Jíří; Otwinowski, Zbyszek; Pokorná, Jana; Plešek, Jaromír; Grüner, Bohumír; Dolečková-Marešová, Lucie; Máša, Martin; Sedláček, Juraj; Bodem, Jochen; Kräusslich, Hans-Georg; Král, Vladimír; Konvalinka, Jan

2005-01-01

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3′ subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition. PMID:16227435

Ebselen: Mechanisms of Glutamate Dehydrogenase and Glutaminase Enzyme Inhibition.

PubMed

Yu, Yan; Jin, Yanhong; Zhou, Jie; Ruan, Haoqiang; Zhao, Han; Lu, Shiying; Zhang, Yue; Li, Di; Ji, Xiaoyun; Ruan, Benfang Helen

2017-12-15

Ebselen modulates target proteins through redox reactions with selenocysteine/cysteine residues, or through binding to the zinc finger domains. However, a recent contradiction in ebselen inhibition of kidney type glutaminase (KGA) stimulated our interest in investigating its inhibition mechanism with glutamate dehydrogenase (GDH), KGA, thioredoxin reductase (TrxR), and glutathione S-transferase. Fluorescein- or biotin-labeled ebselen derivatives were synthesized for mechanistic analyses. Biomolecular interaction analyses showed that only GDH, KGA, and TrxR proteins can bind to the ebselen derivative, and the binding to GDH and KGA could be competed off by glutamine or glutamate. From the gel shift assays, the fluorescein-labeled ebselen derivative could co-migrate with hexameric GDH and monomeric/dimeric TrxR in a dose-dependent manner; it also co-migrated with KGA but disrupted the tetrameric form of the KGA enzyme at a high compound concentration. Further proteomic analysis demonstrated that the ebselen derivative could cross-link with proteins through a specific cysteine at the active site of GDH and TrxR proteins, but for KGA protein, the binding site is at the N-terminal appendix domain outside of the catalytic domain, which might explain why ebselen is not a potent KGA enzyme inhibitor in functional assays. In conclusion, ebselen could inhibit enzyme activity by binding to the catalytic domain or disruption of the protein complex. In addition, ebselen is a relatively potent selective GDH inhibitor that might provide potential therapeutic opportunities for hyperinsulinism-hyperammonemia syndrome patients who have the mutational loss of GTP inhibition.

Antigen-binding thymus-derived lymphocytes

PubMed Central

Hogg, Nancy M.; Greaves, M. F.

1972-01-01

Thymus-derived `rosette'-forming lymphocytes which have been separated from other SRBC-sensitive cells by means of cotton wool columns were examined for the presence of immunoglobulin. This was carried out by inhibition of rosette formation by anti-immunoglobulin sera. Inhibition was effected by a number of anti-IgM sera shown to contain antibodies with specificities directed towards the `hinge' region of the μ chain. No other heavy chain specific antisera were inhibitory. The ratio of rosette inhibition by anti-κ and anti-λ light chain sera varied during the course of the response to SRBC, the latter inhibiting by 89 per cent 3 days post-immunization. PMID:4113387

Vaccine-Induced Plasma IgA Specific for the C1 Region of the HIV-1 Envelope Blocks Binding and Effector Function of IgG

DTIC Science & Technology

2013-05-28

uninfected vaccine recipients in RV144. Moreover, Env-specific IgA antibodies from RV144 vaccinees blocked the binding of ADCC-mediating mAb to HIV-1 Env... vaccine re- cipients in the case control study. There was a significantly greater number of infected vaccinees with IgA/IgG ratio >1e-02 (A1 Congp140 Env... vaccine efficacy. Second, we demonstrated that IgA mAbs isolated from RV144 vaccinees can both inhibit Env binding and block ADCC function of vaccine

Bile Salt-Stimulated Lipase from Human Milk Binds DC-SIGN and Inhibits Human Immunodeficiency Virus Type 1 Transfer to CD4+ T Cells

PubMed Central

Naarding, Marloes A.; Dirac, Annette M.; Ludwig, Irene S.; Speijer, Dave; Lindquist, Susanne; Vestman, Eva-Lotta; Stax, Martijn J.; Geijtenbeek, Teunis B. H.; Pollakis, Georgios; Hernell, Olle; Paxton, William A.

2006-01-01

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents. PMID:17005819

[Targeting of membrane receptor tyrosine kinases: is there resistance in the HER?].

PubMed

Monnier, Lucile; Milano, Gérard; Penault-Llorca, Frédérique; Merlin, Jean-Louis

2004-09-01

Human Epidermal growth factor Receptors (HER) play an important role in cellular proliferation, and differentiation. Their overexpression in tumor tissues is often associated with a poor prognosis. Consequently, HER receptors are interesting therapeutic targets for cancer treatment. Two strategies are proposed. First, monoclonal antibodies can be used to inhibit the binding of one ligand to its receptor. The second approach is based upon the designing of tyrosine kinase inhibitors capable to bind into the phosphorylation site of the receptor. Consequently, both approaches block the signal transduction downstream. Resistance to anti receptor tyrosine kinase therapy can lead to enhanced morbidity associated with high therapeutic cost. Different mechanisms can be implicated. Non specific mechanisms include alterations of the signal transduction pathways (PI3K/AKT), recruitment of alternative receptor tyrosine kinase pathways (IGFR, VEGFR) and proteasome degradation inhibition. Other mechanisms are specific to HER and rely on inhibition of the binding of monoclonal antibodies (sialomucin-MUC4), heterodimerisation of HER, truncated soluble receptors intervention and mutated variants, as demonstrated very recently with EGF receptors, or genetic polymorphism. This paper reviews these different resistance mechanisms that have been identified in preclinical and clinical situations.

Ca2+ channel blockers interact with alpha 2-adrenergic receptors in rabbit ileum.

PubMed

Homaidan, F R; Donowitz, M; Wicks, J; Cusolito, S; el Sabban, M E; Weiland, G A; Sharp, W G

1988-04-01

An interaction between Ca2+ channel blockers and alpha 2-adrenergic receptors has been demonstrated in rabbit ileum by studying the effect of clonidine on active electrolyte transport, under short-circuited conditions, in the presence and absence of several Ca2+ channel blocking agents. Clonidine, verapamil, diltiazem, cadmium, and nitrendipine all decrease short-circuit current and stimulate NaCl absorption to different extents with clonidine having the largest effect. Exposure to verapamil, diltiazem, and cadmium inhibited the effects of clonidine on transport, whereas nitrendipine had no such effect. Verapamil, diltiazem, and cadmium, but not nitrendipine, also decreased the specific binding of [3H]alpha 2-adrenergic agents to a preparation of ileal basolateral membranes explaining the observed decrease in the transport effects of clonidine. The effective concentrations of the Ca2+ channel blockers that inhibited the effects of clonidine on transport were fairly similar to the concentrations needed to inhibit its specific binding. The displacement of clonidine by calcium channel blockers is ascribed to a nonspecific effect of these agents, although the possibility that their effects are exerted via their binding to the calcium channels is not excluded.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vermaas, Josh V.; Petridis, Loukas; Qi, Xianghong

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process. By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose bindingmore » of TrCel7A (Y466, Y492, and Y493). In conclusion, lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.« less

Botulinum Neurotoxins and Botulism: A Novel Therapeutic Approach

PubMed Central

Thanongsaksrikul, Jeeraphong; Chaicumpa, Wanpen

2011-01-01

Specific treatment is not available for human botulism. Current remedial mainstay is the passive administration of polyclonal antibody to botulinum neurotoxin (BoNT) derived from heterologous species (immunized animal or mouse hybridoma) together with supportive and symptomatic management. The antibody works extracellularly, probably by blocking the binding of receptor binding (R) domain to the neuronal receptors; thus inhibiting cellular entry of the holo-BoNT. The antibody cannot neutralize the intracellular toxin. Moreover, a conventional antibody with relatively large molecular size (150 kDa) is not accessible to the enzymatic groove and, thus, cannot directly inhibit the BoNT zinc metalloprotease activity. Recently, a 15–20 kDa single domain antibody (VHH) that binds specifically to light chain of BoNT serotype A was produced from a humanized-camel VH/VHH phage display library. The VHH has high sequence homology (>80%) to the human VH and could block the enzymatic activity of the BoNT. Molecular docking revealed not only the interface binding between the VHH and the toxin but also an insertion of the VHH CDR3 into the toxin enzymatic pocket. It is envisaged that, by molecular linking the VHH to a cell penetrating peptide (CPP), the CPP-VHH fusion protein would be able to traverse the hydrophobic cell membrane into the cytoplasm and inhibit the intracellular BoNT. This presents a novel and safe immunotherapeutic strategy for botulism by using a cell penetrating, humanized-single domain antibody that inhibits the BoNT by means of a direct blockade of the groove of the menace enzyme. PMID:22069720

Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morrison, W.J.; Offner, H.; Vandenbark, A.A.

1989-01-01

The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphoctyes. Saturation binding by monosialylated ({sub 3}H)-GM{sub 1} to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (K{sub D}) of 2.2 {plus minus} 1.4 {mu}M and a binding capacity near 2 fmoles/cell. Competitive inhibition of ({sup 3}H)- GM{sub 1} binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM{sub 1}-congeneric gangliosides. A comparison between the results of these binding studies and gangliosides-induced decrease of CD4 expression demonstrated that every aspect of ({supmore » 3}H)-GM{sub 1} binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of gangliosides binding to specific sites on CD4{sup {double dagger}} T-helper lymphocytes.« less

Receptor binding sites for atrial natriuretic factor are expressed by brown adipose tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bacay, A.C.; Mantyh, C.R.; Vigna, S.R.

1988-09-01

To explore the possibility that atrial natriuretic factor (ANF) is involved in thermoregulation we used quantitative receptor autoradiography and homogenate receptor binding assays to identify ANF bindings sites in neonatal rat and sheep brown adipose tissue, respectively. Using quantitative receptor autoradiography were were able to localize high levels of specific binding sites for {sup 125}I-rat ANF in neonatal rat brown adipose tissue. Homogenate binding assays on sheep brown fat demonstrated that the radioligand was binding to the membrane fraction and that the specific binding was not due to a lipophilic interaction between {sup 125}I-rat ANF and brown fat. Specific bindingmore » of {sup 125}I-rat ANF to the membranes of brown fat cells was inhibited by unlabeled rat ANF with a Ki of 8.0 x 10(-9) M, but not by unrelated peptides. These studies demonstrate that brown fat cells express high levels of ANF receptor binding sites in neonatal rat and sheep and suggest that ANF may play a role in thermoregulation.« less

High doses of recombinant mannan-binding lectin inhibit the binding of influenza A(H1N1)pdm09 virus with cells expressing DC-SIGN.

PubMed

Yu, Lei; Shang, Shiqiang; Tao, Ran; Wang, Caiyun; Zhang, Li; Peng, Hao; Chen, Yinghu

2017-07-01

The pandemic influenza A (H1N1)pdm09 virus continues to be a threat to human health. Low doses of mannan-binding lectin (MBL) (<1 μg/mL) were shown not to protect against influenza A(H1N1)pdm09 infection. However, the effect of high doses of MBL has not been investigated. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) has been proposed as an alternative receptor for influenza A(H1N1)pdm09 virus. In this study, we examined the expression of DC-SIGN on DCs as well as on acute monocytic leukemia cell line, THP-1. High doses of recombinant or human MBL inhibited binding of influenza A(H1N1)pdm09 to both these cell types in the presence of complement derived from bovine serum. Further, anti-DC-SIGN monoclonal antibody inhibited binding of influenza A(H1N1)pdm09 to both DC-SIGN-expressing DCs and THP-1 cells. This study demonstrates that high doses of MBL can inhibit binding of influenza A(H1N1)pdm09 virus to DC-SIGN-expressing cells in the presence of complement. Our results suggest that DC-SIGN may be an alternative receptor for influenza A(H1N1)pdm09 virus. © 2017 APMIS. Published by John Wiley & Sons Ltd.

Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication.

PubMed

Peixoto, Paul; Liu, Yang; Depauw, Sabine; Hildebrand, Marie-Paule; Boykin, David W; Bailly, Christian; Wilson, W David; David-Cordonnier, Marie-Hélène

2008-06-01

The development of small molecules to control gene expression could be the spearhead of future-targeted therapeutic approaches in multiple pathologies. Among heterocyclic dications developed with this aim, a phenyl-furan-benzimidazole dication DB293 binds AT-rich sites as a monomer and 5'-ATGA sequence as a stacked dimer, both in the minor groove. Here, we used a protein/DNA array approach to evaluate the ability of DB293 to specifically inhibit transcription factors DNA-binding in a single-step, competitive mode. DB293 inhibits two POU-domain transcription factors Pit-1 and Brn-3 but not IRF-1, despite the presence of an ATGA and AT-rich sites within all three consensus sequences. EMSA, DNase I footprinting and surface-plasmon-resonance experiments determined the precise binding site, affinity and stoichiometry of DB293 interaction to the consensus targets. Binding of DB293 occurred as a cooperative dimer on the ATGA part of Brn-3 site but as two monomers on AT-rich sites of IRF-1 sequence. For Pit-1 site, ATGA or AT-rich mutated sequences identified the contribution of both sites for DB293 recognition. In conclusion, DB293 is a strong inhibitor of two POU-domain transcription factors through a cooperative binding to ATGA. These findings are the first to show that heterocyclic dications can inhibit major groove transcription factors and they open the door to the control of transcription factors activity by those compounds.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beaumont, K.; Vaughn, D.A.; Fanestil, D.D.

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. The authors report here that ({sup 3}H)metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. ({sup 3}H)Metolazone bound with high affinity to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or not binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated withmore » their clinical potency. Halide anions specifically inhibited high-affinity binding of ({sup 3}H)metolazone to this site. ({sup 3})Metolazone also bound with lower affinity to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had K{sub i} values of 0.6-10 {mu}M for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity ({sup 3}H)metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.« less

Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure

PubMed Central

Nicklisch, Sascha C. T.; Rees, Steven D.; McGrath, Aaron P.; Gökirmak, Tufan; Bonito, Lindsay T.; Vermeer, Lydia M.; Cregger, Cristina; Loewen, Greg; Sandin, Stuart; Chang, Geoffrey; Hamdoun, Amro

2016-01-01

The world’s oceans are a global reservoir of persistent organic pollutants to which humans and other animals are exposed. Although it is well known that these pollutants are potentially hazardous to human and environmental health, their impacts remain incompletely understood. We examined how persistent organic pollutants interact with the drug efflux transporter P-glycoprotein (P-gp), an evolutionarily conserved defense protein that is essential for protection against environmental toxicants. We identified specific congeners of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers that inhibit mouse and human P-gp, and determined their environmental levels in yellowfin tuna from the Gulf of Mexico. In addition, we solved the cocrystal structure of P-gp bound to one of these inhibitory pollutants, PBDE (polybrominated diphenyl ether)–100, providing the first view of pollutant binding to a drug transporter. The results demonstrate the potential for specific binding and inhibition of mammalian P-gp by ubiquitous congeners of persistent organic pollutants present in fish and other foods, and argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals. PMID:27152359

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions.

PubMed

Srikant, C B; Dahan, A; Craig, C

1990-02-04

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2+, Mn2+, Ca2+ and Co2+ augmented the binding of both T* SS-14 and LTT* SS-28, while higher than 4 mM Co2+ inhibited binding of both ligands. LTT* SS-28 binding was reduced in the presence of high concentrations of Ba2+ and Mn2+ also. Interestingly Ca2+ at higher than 10 mM preferentially inhibited LTT* SS-28 binding and increased the affinity of SS-14 but not SS-28 for LTT* SS-28 binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific Binding of Adamantane Drugs and Direction of their Polar Amines in the Pore of the Influenza M2 Transmembrane Domain in Lipid Bilayers and Dodecylphosphocholine Micelles Determined by NMR Spectroscopy

PubMed Central

Cady, Sarah D.; Wang, Jun; Wu, Yibing; DeGrado, William F.; Hong, Mei

2011-01-01

The transmembrane domain of the influenza M2 protein (M2TM) forms a tetrameric proton channel important for the virus lifecycle. The proton-channel activity is inhibited by amine-containing adamantyl drugs amantadine and rimantadine, which have been shown to bind specifically to the pore of M2TM near Ser31. However, whether the polar amine points to the N- or C-terminus of the channel has not yet been determined. Elucidating the polar group direction will shed light on the mechanism by which drug binding inhibits this proton channel and will facilitate rational design of new inhibitors. In this study, we determine the polar amine direction using M2TM reconstituted in lipid bilayers as well as DPC micelles. 13C-2H rotational-echo double-resonance NMR experiments of 13C-labeled M2TM and methyl-deuterated rimantadine in lipid bilayers showed that the polar amine pointed to the C-terminus of the channel, with the methyl group close to Gly34. Solution NMR experiments of M2TM in dodecylphosphocholine (DPC) micelles indicate that drug binding causes significant chemical shift perturbations of the protein that are very similar to those seen for M2TM and M2(18–60) bound to lipid bilayers. Specific 2H-labeling of the drugs permitted the assignment of drug-protein cross peaks, which indicate that amantadine and rimantadine bind to the pore in the same fashion as for bilayer-bound M2TM. These results strongly suggest that adamantyl inhibition of M2TM is achieved not only by direct physical occlusion of the pore but also by perturbing the equilibrium constant of the proton-sensing residue His37. The reproduction of the pharmacologically relevant specific pore-binding site in DPC micelles, which was not observed with a different detergent, DHPC, underscores the significant influence of the detergent environment on the functional structure of membrane proteins. PMID:21381693

A small-molecule compound inhibits a collagen-specific molecular chaperone and could represent a potential remedy for fibrosis.

PubMed

Ito, Shinya; Ogawa, Koji; Takeuchi, Koh; Takagi, Motoki; Yoshida, Masahito; Hirokawa, Takatsugu; Hirayama, Shoshiro; Shin-Ya, Kazuo; Shimada, Ichio; Doi, Takayuki; Goshima, Naoki; Natsume, Tohru; Nagata, Kazuhiro

2017-12-08

Fibrosis can disrupt tissue structure and integrity and impair organ function. Fibrosis is characterized by abnormal collagen accumulation in the extracellular matrix. Pharmacological inhibition of collagen secretion therefore represents a promising strategy for the management of fibrotic disorders, such as liver and lung fibrosis. Hsp47 is an endoplasmic reticulum (ER)-resident collagen-specific molecular chaperone essential for correct folding of procollagen in the ER. Genetic deletion of Hsp47 or inhibition of its interaction with procollagen interferes with procollagen triple helix production, which vastly reduces procollagen secretion from fibroblasts. Thus, Hsp47 could be a potential and promising target for the management of fibrosis. In this study, we screened small-molecule compounds that inhibit the interaction of Hsp47 with collagen from chemical libraries using surface plasmon resonance (BIAcore), and we found a molecule AK778 and its cleavage product Col003 competitively inhibited the interaction and caused the inhibition of collagen secretion by destabilizing the collagen triple helix. Structural information obtained with NMR analysis revealed that Col003 competitively binds to the collagen-binding site on Hsp47. We propose that these structural insights could provide a basis for designing more effective therapeutic drugs for managing fibrosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements.

PubMed

Liu, Shu; Bolger, Joshua K; Kirkland, Lindsay O; Premnath, Padmavathy N; McInnes, Campbell

2010-12-17

An alternative strategy for inhibition of the cyclin dependent kinases (CDKs) in antitumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation, and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential to generate highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anticancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27(KIP)¹, with cyclin D1. This information has been used to shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 that can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and nonpeptidic compounds have been synthesized in order to explore structure-activity relationship for binding to the cyclin D1 groove, which to date has not been carried out in a systematic fashion. Collectively, the data presented provide new insights into how compounds can be developed that function as chemical biology probes to determine the cellular and antitumor effects of CDK inhibition. Furthermore, such compounds will serve as templates for structure-guided efforts to develop potential therapeutics based on selective inhibition of CDK4/cyclin D activity.

Effects of calcium and magnesium ions on the interaction of corticosterone with rat brain cytosol receptor(s).

PubMed

Nakai, T; Ueda, M; Takeda, R

1978-01-01

The apparent maximum corticosterone binding (B max) with rat brain cytosol and the apparent dissociation constant of this steroid-receptor binding (Kd) estimated with a Scatchard plot was 2.9 X 10(-13) moles/mg cytosol protein and 4.0 X 10(-9) M, respectively. When increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, a specific [3H] corticosterone binding increased 4-fold by CaCl2 at concentrations of 1.0-2.0 mM and 1.5-fold by MgCl2 at concentrations of 0.5-5.0 mM. The addition of MnCl2 and KCl did not affect this binding. Binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EGTA and complete inhibition was observed at concentrations equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.

Effect of SR-49059, a vasopressin V1a antagonist, on human vascular smooth muscle cells.

PubMed

Serradeil-Le Gal, C; Herbert, J M; Delisee, C; Schaeffer, P; Raufaste, D; Garcia, C; Dol, F; Marty, E; Maffrand, J P; Le Fur, G

1995-01-01

The effects of SR-49059, a new nonpeptide and selective arginine vasopressin (AVP) V1a antagonist, were investigated in binding and functional studies on cultured human aortic vascular smooth muscle cells (VSMC). Characterization of human vascular V1a receptors, using a specific V1a radioiodinated ligand, showed that [125I]-linear AVP antagonist binding to human VSMC membranes was time dependent, reversible, and saturable. A single population of high-affinity binding sites (apparent equilibrium dissociation constant = 15 +/- 6 pM; maximum binding density = 36 +/- 5 fmol/mg protein, i.e., approximately 3,000 sites/cell) with the expected V1a profile was identified. Exposure of these cells to AVP dose-dependently produced cytosolic free [Ca2+] increase [AVP concentration required to obtain a half-maximal response (EC50) = 23 +/- 9 nM] and proliferation (EC50 = 3.2 +/- 0.5 nM). SR-49059 strongly and stereospecifically inhibited [125I]-linear AVP antagonist binding to VSMC V1a receptors [inhibition constant (Ki) = 1.4 +/- 0.3 nM], AVP-evoked Ca2+ increase [concentration of inhibitor required to obtain 50% inhibition of specific binding (IC50) = 0.41 +/- 0.06 nM], and the mitogenic effects induced by 100 nM AVP (IC50 = 0.83 +/- 0.04 nM). OPC-21268, another nonpeptide V1a antagonist, was more than two orders of magnitude less potent than SR-49059 in these models. However, the consistent affinity (Ki = 138 +/- 21 nM) and activity found with OPC-21268 on human VSMC in comparison with the inactivity already observed for other human V1a receptors (liver, platelets, adrenals, and uterus) strongly suggested the existence of human AVP V1a-receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

USP7 small-molecule inhibitors interfere with ubiquitin binding.

PubMed

Kategaya, Lorna; Di Lello, Paola; Rougé, Lionel; Pastor, Richard; Clark, Kevin R; Drummond, Jason; Kleinheinz, Tracy; Lin, Eva; Upton, John-Paul; Prakash, Sumit; Heideker, Johanna; McCleland, Mark; Ritorto, Maria Stella; Alessi, Dario R; Trost, Matthias; Bainbridge, Travis W; Kwok, Michael C M; Ma, Taylur P; Stiffler, Zachary; Brasher, Bradley; Tang, Yinyan; Jaishankar, Priyadarshini; Hearn, Brian R; Renslo, Adam R; Arkin, Michelle R; Cohen, Frederick; Yu, Kebing; Peale, Frank; Gnad, Florian; Chang, Matthew T; Klijn, Christiaan; Blackwood, Elizabeth; Martin, Scott E; Forrest, William F; Ernst, James A; Ndubaku, Chudi; Wang, Xiaojing; Beresini, Maureen H; Tsui, Vickie; Schwerdtfeger, Carsten; Blake, Robert A; Murray, Jeremy; Maurer, Till; Wertz, Ingrid E

2017-10-26

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Lithocholic acid is an endogenous inhibitor of MDM4 and MDM2

PubMed Central

Vogel, Simon M.; Bauer, Matthias R.; Joerger, Andreas C.; Wilcken, Rainer; Brandt, Tobias; Veprintsev, Dmitry B.; Rutherford, Trevor J.; Fersht, Alan R.; Boeckler, Frank M.

2012-01-01

The proteins MDM2 and MDM4 are key negative regulators of the tumor suppressor protein p53, which are frequently upregulated in cancer cells. They inhibit the transactivation activity of p53 by binding separately or in concert to its transactivation domain. MDM2 is also a ubiquitin ligase that leads to the degradation of p53. Accordingly, MDM2 and MDM4 are important targets for drugs to inhibit their binding to p53. We found from in silico screening and confirmed by experiment that lithocholic acid (LCA) binds to the p53 binding sites of both MDM2 and MDM4 with a fivefold preference for MDM4. LCA is an endogenous steroidal bile acid, variously reported to have both carcinogenic and apoptotic activities. The comparison of LCA effects on apoptosis in HCT116 p53+/+ vs. p53-/- cells shows a predominantly p53-mediated induction of caspase-3/7. The dissociation constants are in the μM region, but only modest inhibition of binding of MDM2 and MDM4 is required to negate their upregulation because they have to compete with transcriptional coactivator p300 for binding to p53. Binding was weakened by structural changes in LCA, and so it may be a natural ligand of MDM2 and MDM4, raising the possibility that MDM proteins may be sensors for specific steroids. PMID:23035244

An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basu, Niladri; Department of Natural Resource Sciences, McGill University, Ste.-Anne-de-Bellevue, Quebec, H9X 3V9; Stamler, Christopher J.

2005-05-15

Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl{sub 2}) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl{sub 2} and MeHg on [{sup 3}H]-quinuclidinyl benzilate ([{sup 3}H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse,more » mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B {sub max}) and ligand affinity (K {sub d}). Subsequently, samples were exposed to HgCl{sub 2} or MeHg to derive IC50 values and inhibition constants (K {sub i}). Results demonstrate that HgCl{sub 2} is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [{sup 3}H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.« less

Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

PubMed

Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

2003-08-01

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

Site-specific effects of the nonsteroidal anti-inflammatory drug lysine clonixinate on rat brain opioid receptors.

PubMed

Ortí, E; Coirini, H; Pico, J C

1999-04-01

In addition to effects in the periphery through inhibition of prostaglandin synthesis, several lines of evidence suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) act in the central nervous system. The possibility that the central action of NSAIDs involves regulation of opioid receptors was investigated by quantitative autoradiography of mu, delta, and kappa sites in rat brain slices. Increased (p < 0.05) labeling of mu receptors was observed in thalamic nuclei, gyrus dentate, and layers of the parietal cortex of rats treated for 10 days with lysine clonixinate. Labeling of delta receptors was lower in the lateral septum, and kappa sites decreased in thalamic nuclei. These effects were not mediated through direct interaction with opioid-binding sites, since receptor-binding assays using rat brain membranes confirmed that clonixinate up to 1 x 10(-4) mol/l does not inhibit mu, delta, and kappa receptor specific binding. Central effects of NSAIDs might, therefore, involve interaction with the opioid receptor system through indirect mechanisms.

98 Specific IGE and IGG Binding to Allergoids of Phleum pratense

PubMed Central

Cases, Barbara; Fernandez-Caldas, Enrique; Tudela, Jose Ignacio; Fernandez, Eva Abel; Sanchez-Garcia, Silvia; Ibañez, M. Dolores; Escudero, Carmelo; Casanovas, Miguel

2012-01-01

Background Allergoids were first used in the decades of the 60s and 70s of the last century as an effective treatment of allergic respiratory diseases. Allergoids can be modified with formaldehyde or glutaraldehyde. Modified allergens, or allergoids, decrease the risk of adverse reactions while administering higher allergen doses. The objective of this study was to analyse specific IgE and IgG binding to glutaraldehyde modified and non-modified allergen extracts of Phleum pratense. Methods The sera of 69 patients sensitized to P. pratense were tested. All these patients had signs and symptoms of rhinoconjunctivitis with, or without, asthma in May and June of 2011. All these patients had positive skin prick tests to a standardized extract of P. pratense, and other grass species. Most patients were also sensitized to olive pollen. Specific IgE and IgG binding were analysed by direct ELISA against P. pratense native (non-modified) and allergoid extracts. Relative potencies were evaluated through ELISA inhibition assays, and the protein composition of non-modified and allergoid samples was determined by Mass Spectrometry (MS/MS). Results Mean Specific IgE levels against the native extract was 16.68 ± 11.65 Units (U) and against the allergoid: 7.26 ± 8.24 U (P < 0.0001; Mann-Whitney). On the other hand, mean specific IgG binding against the non-modified extract was 90.34 ± 75.57 U versus 76.19 ± 70.31 U against the allergoid (P = 0.16; Mann-Whitney). Linear regression coefficients obtained between immunoglobulin reactivity against both extracts were: r2 = 0.51 for specific IgE and r2 = 0.83 for specific IgG. An important decrease in the allergenic activity, measured by inhibition ELISA, was clearly observed. The MS/MS assay revealed the presence of the mayor allergen, and some isoforms, in non-modified and allergoid extracts. Conclusions Results obtained demonstrate that the glutaraldehyde polymerization process induces an important decrease in specific IgE binding to allergoids of P. pratense while there are no significant differences in specific IgG binding. The allergenic composition of the P. pratense allergoid was equivalent to the non-modified pollen extract.

Targeting the disordered C-terminus of PTP1B with an allosteric inhibitor

PubMed Central

Krishnan, Navasona; Koveal, Dorothy; Miller, Daniel H.; Xue, Bin; Akshinthala, Sai Dipikaa; Kragelj, Jaka; Jensen, Malene Ringkjøbing; Gauss, Carla-Maria; Page, Rebecca; Blackledge, Martin; Muthuswamy, Senthil K.; Peti, Wolfgang; Tonks, Nicholas K.

2014-01-01

PTP1B, a validated therapeutic target for diabetes and obesity, plays a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We defined a novel mechanism of allosteric inhibition that targets the C-terminal, non-catalytic segment of PTP1B. We present the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small molecule inhibitor, MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules. PMID:24845231

Interactions of Monoamine Oxidases with the Antiepileptic Drug Zonisamide: Specificity of Inhibition and Structure of the Human Monoamine oxidase B Complex

PubMed Central

Binda, Claudia; Aldeco, Milagros; Mattevi, Andrea; Edmondson, Dale E.

2010-01-01

The binding of zonisamide to purified, recombinant monoamine oxidases (MAOs) has been investigated. It is a competitive inhibitor of human MAO B (Ki = 3.1 ± 0.3 μM), of rat MAO B (Ki = 2.9 ± 0.5 μM), and of zebrafish MAO (Ki = 30.8 ± 5.3 μM). No inhibition is observed with purified human or rat MAO A. The 1.8 Å structure of the MAO B complex demonstrates that it binds within the substrate cavity. PMID:21175212

Binding of Soluble Yeast β-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

PubMed Central

Bose, Nandita; Chan, Anissa S. H.; Guerrero, Faimola; Maristany, Carolyn M.; Qiu, Xiaohong; Walsh, Richard M.; Ertelt, Kathleen E.; Jonas, Adria Bykowski; Gorden, Keith B.; Dudney, Christine M.; Wurst, Lindsay R.; Danielson, Michael E.; Elmasry, Natalie; Magee, Andrew S.; Patchen, Myra L.; Vasilakos, John P.

2013-01-01

The immunomodulatory properties of yeast β-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate β-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble β-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast β-glucan, this study evaluated and characterized the binding of soluble β-glucan to human neutrophils and monocytes. The results demonstrated that soluble β-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble β-glucan in these cells. Binding of soluble β-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble β-glucan was demonstrated by detection of iC3b, the complement opsonin on β-glucan-bound cells, as well as by the direct binding of iC3b to β-glucan in the absence of cells. Binding of β-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding. PMID:23964276

Fast and automated functional classification with MED-SuMo: an application on purine-binding proteins.

PubMed

Doppelt-Azeroual, Olivia; Delfaud, François; Moriaud, Fabrice; de Brevern, Alexandre G

2010-04-01

Ligand-protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED-SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED-SMA is an automated and fast method to classify binding sites. It is based on MED-SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora-A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED-SMA approach is demonstrated as it gathers binding sites of proteins with similar structure-activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target-based drug design and prediction of cross-reactivity and therefore potential toxic side effects.

Fast and automated functional classification with MED-SuMo: An application on purine-binding proteins

PubMed Central

Doppelt-Azeroual, Olivia; Delfaud, François; Moriaud, Fabrice; de Brevern, Alexandre G

2010-01-01

Ligand–protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED-SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED-SMA is an automated and fast method to classify binding sites. It is based on MED-SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora-A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED-SMA approach is demonstrated as it gathers binding sites of proteins with similar structure-activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target-based drug design and prediction of cross-reactivity and therefore potential toxic side effects. PMID:20162627

Stapled peptide inhibitors of RAB25 target context-specific phenotypes in cancer | Office of Cancer Genomics

Cancer.gov

Recent evidence has established a role for the small GTPase RAB25, as well as related effector proteins, in enacting both pro-oncogenic and anti-oncogenic phenotypes in specific cellular contexts. Here we report the development of all-hydrocarbon stabilized peptides derived from the RAB-binding FIP-family of proteins to target RAB25. Relative to unmodified peptides, optimized stapled peptides exhibit increased structural stability, binding affinity, cell permeability, and inhibition of RAB25:FIP complex formation.

A designed inhibitor of a CLC antiporter blocks function through a unique binding mode

PubMed Central

Howery, Andrew E.; Elvington, Shelley; Abraham, Sherwin J.; Choi, Kee-Hyun; Phillips, Sabrina; Ryan, Christopher M.; Sanford, R. Lea; Simpson-Dworschak, Sierra; Almqvist, Jonas; Tran, Kevin; Chew, Thomas A.; Zachariae, Ulrich; Andersen, Olaf S.; Whitelegge, Julian; Matulef, Kimberly; Du Bois, Justin; Maduke, Merritt C.

2012-01-01

SUMMARY The lack of small-molecule inhibitors for anion-selective transporters and channels has impeded our understanding of the complex mechanisms that underlie ion passage. The ubiquitous CLC “Chloride Channel” family represents a unique target for biophysical and biochemical studies because its distinctive protein fold supports both passive chloride channels and secondary-active chloride-proton transporters. Here, we describe the synthesis and characterization of the first specific small-molecule inhibitor directed against a CLC antiporter (ClC-ec1). This compound, 4,4′-octanamidostilbene-2,2′-disulfonate (OADS), inhibits ClC-ec1 with low micromolar affinity and has no specific effect on a CLC channel (ClC-1). Inhibition of ClC-ec1 occurs by binding to two distinct intracellular sites. The location of these sites and the lipid-dependence of inhibition suggest potential mechanisms of action. The discovery of this compound will empower research to elucidate differences between antiporter and channel mechanisms and to develop treatments for CLC-mediated disorders. PMID:23177200

Screening and Development of New Inhibitors of FtsZ from M. Tuberculosis

PubMed Central

Mathew, Bini; Ross, Larry; Connelly, Michele C.; Lofton, Hava; Rajagopalan, Malini; Guy, R. Kiplin; Reynolds, Robert C.

2016-01-01

A variety of commercial analogs and a newer series of Sulindac derivatives were screened for inhibition of M. tuberculosis (Mtb) in vitro and specifically as inhibitors of the essential mycobacterial tubulin homolog, FtsZ. Due to the ease of preparing diverse analogs and a favorable in vivo pharmacokinetic and toxicity profile of a representative analog, the Sulindac scaffold may be useful for further development against Mtb with respect to in vitro bacterial growth inhibition and selective activity for Mtb FtsZ versus mammalian tubulin. Further discovery efforts will require separating reported mammalian cell activity from both antibacterial activity and inhibition of Mtb FtsZ. Modeling studies suggest that these analogs bind in a specific region of the Mtb FtsZ polymer that differs from human tubulin and, in combination with a pharmacophore model presented herein, future hybrid analogs of the reported active molecules that more efficiently bind in this pocket may improve antibacterial activity while improving other drug characteristics. PMID:27768711

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B.

PubMed

Koch, Alexander W; Mathivet, Thomas; Larrivée, Bruno; Tong, Raymond K; Kowalski, Joe; Pibouin-Fragner, Laurence; Bouvrée, Karine; Stawicki, Scott; Nicholes, Katrina; Rathore, Nisha; Scales, Suzie J; Luis, Elizabeth; del Toro, Raquel; Freitas, Catarina; Bréant, Christiane; Michaud, Annie; Corvol, Pierre; Thomas, Jean-Léon; Wu, Yan; Peale, Franklin; Watts, Ryan J; Tessier-Lavigne, Marc; Bagri, Anil; Eichmann, Anne

2011-01-18

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature. Copyright © 2011 Elsevier Inc. All rights reserved.

Triggers of key calcium signals during erythrocyte invasion by Plasmodium falciparum

PubMed Central

Gao, Xiaohong; Gunalan, Karthigayan; Yap, Sally Shu Lin; Preiser, Peter R.

2013-01-01

Invasion of erythrocytes by Plasmodium falciparum merozoites is a complex multi-step process mediated by specific interactions between host receptors and parasite ligands. Reticulocyte-binding protein homologues (RHs) and erythrocyte-binding-like (EBL) proteins are discharged from specialized organelles and used in early steps of invasion. Here we show that monoclonal antibodies against PfRH1 (an RH) block merozoite invasion by specifically inhibiting calcium signalling in the parasite, whereas invasion-inhibiting monoclonal antibodies targeting EBA175 (an EBL protein) have no effect on signalling. We further show that inhibition of this calcium signalling prevents EBA175 discharge and thereby formation of the junction between parasite and host cell. Our results indicate that PfRH1 has an initial sensing as well as signal transduction role that leads to the subsequent release of EBA175. They also provide new insights on how RH–host cell interactions lead to essential downstream signalling events in the parasite, suggesting new targets for malaria intervention. PMID:24280897

Differences in the distribution and characteristics of tachykinin NK1 binding sites between human and guinea pig lung.

PubMed Central

Walsh, D A; Salmon, M; Featherstone, R; Wharton, J; Church, M K; Polak, J M

1994-01-01

1. The distribution and characteristics of tachykinin NK1 binding sites have been compared in human and guinea pig lung using quantitative in vitro receptor autoradiography with [125I]-Bolton Hunter-labelled substance P ([125I]-BH-SP). In addition, the effects on these sites of ovalbumin sensitization and challenge have been determined in guinea pig lung. 2. [125I]-BH-SP bound specifically and with high affinity to microvascular endothelium in both human and guinea pig lung, but to bronchial smooth muscle and pulmonary artery media in only guinea pig lung. 3. Specific binding of [125I]-BH-SP to guinea pig bronchial smooth muscle was positively correlated with airway diameter in the range 150-800 microns and was less dense in trachea than in main bronchi. 4. [125I]-BH-SP binding was inhibited by tachykinins with rank orders of affinity of SP > NKA > NKB (human microvessels) and SP > NKA = NKB (guinea pig bronchi and pulmonary arteries). NKA displayed a higher affinity for [125I]-BH-SP binding sites in human microvessels than in guinea pig tissues (P < 0.0001), indicating differences in selectivity for tachykinins between human and guinea pig NK1 receptors. 5. In both human and guinea pig lung, [125I]-BH-SP binding was inhibited by the specific tachykinin receptor antagonists FK888 (NK1 selective antagonist) and FK224 (mixed NK1/NK2 antagonist), with FK888 displaying equal affinity to SP and > 500 times higher affinity than FK224. SP, NKA, NKB and FK888 exhibited similar affinities for [125I]-BH-SP binding sites in both guinea pig arteries and bronchi.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 PMID:7534186

Pathogenic immune mechanisms at the neuromuscular synapse: the role of specific antibody-binding epitopes in myasthenia gravis.

PubMed

Huijbers, M G; Lipka, A F; Plomp, J J; Niks, E H; van der Maarel, S M; Verschuuren, J J

2014-01-01

Autoantibodies against three different postsynaptic antigens and one presynaptic antigen at the neuromuscular junction are known to cause myasthenic syndromes. The mechanisms by which these antibodies cause muscle weakness vary from antigenic modulation and complement-mediated membrane damage to inhibition of endogenous ligand binding and blocking of essential protein-protein interactions. These mechanisms are related to the autoantibody titre, specific epitopes on the target proteins and IgG autoantibody subclass. We here review the role of specific autoantibody-binding epitopes in myasthenia gravis, their possible relevance to the pathophysiology of the disease and potential implications of epitope mapping knowledge for new therapeutic strategies. © 2013 The Association for the Publication of the Journal of Internal Medicine.

Comprehensive Interrogation of Natural TALE DNA Binding Modules and Transcriptional Repressor Domains

PubMed Central

Cong, Le; Zhou, Ruhong; Kuo, Yu-chi; Cunniff, Margaret; Zhang, Feng

2012-01-01

Transcription activator-like effectors (TALE) are sequence-specific DNA binding proteins that harbor modular, repetitive DNA binding domains. TALEs have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the TALE toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat variable diresidue (RVD) Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective TALE transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using TALEs. PMID:22828628

Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1

PubMed Central

Burton, Janet L; Xiong, Yong; Solomon, Mark J

2011-01-01

The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APCCdh1 inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APCCdh1 inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APCCdh1 whereas lysine removal from the APC substrate Hsl1 converted it into a potent APCCdh1 inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APCCdh1. PMID:21460798

The RCAN carboxyl end mediates calcineurin docking-dependent inhibition via a site that dictates binding to substrates and regulators

PubMed Central

Martínez-Martínez, Sara; Genescà, Lali; Rodríguez, Antonio; Raya, Alicia; Salichs, Eulàlia; Were, Felipe; López-Maderuelo, María Dolores; Redondo, Juan Miguel; de la Luna, Susana

2009-01-01

Specificity of signaling kinases and phosphatases toward their targets is usually mediated by docking interactions with substrates and regulatory proteins. Here, we characterize the motifs involved in the physical and functional interaction of the phosphatase calcineurin with a group of modulators, the RCAN protein family. Mutation of key residues within the hydrophobic docking-cleft of the calcineurin catalytic domain impairs binding to all human RCAN proteins and to the calcineurin interacting proteins Cabin1 and AKAP79. A valine-rich region within the RCAN carboxyl region is essential for binding to the docking site in calcineurin. Although a peptide containing this sequence compromises NFAT signaling in living cells, it does not inhibit calcineurin catalytic activity directly. Instead, calcineurin catalytic activity is inhibited by a motif at the extreme C-terminal region of RCAN, which acts in cis with the docking motif. Our results therefore indicate that the inhibitory action of RCAN on calcineurin-NFAT signaling results not only from the inhibition of phosphatase activity but also from competition between NFAT and RCAN for binding to the same docking site in calcineurin. Thus, competition by substrates and modulators for a common docking site appears to be an essential mechanism in the regulation of Ca2+-calcineurin signaling. PMID:19332797

Effect of heterogeneity of carcinoembryonic antigen on liver cell membrane binding and its kinetics of removal from circulation.

PubMed

Byrn, R A; Medrek, P; Thomas, P; Jeanloz, R W; Zamcheck, N

1985-07-01

Carcinoembryonic antigen (CEA) is a glycoprotein metabolized primarily by the liver. Subcellular fractions of rat liver were examined for CEA binding activity. Hepatocyte plasma membrane and microsome fractions bound CEA, and this binding shared the calcium requirement, neuraminidase sensitivity, and carbohydrate specificity of the hepatocyte asialoglycoprotein receptor. CEA had previously been shown to react with this galactose-specific receptor, in vivo, only following neuraminidase treatment. Galactose receptor binding of CEA was measured in three different purified CEA preparations. The fraction of CEA capable of binding to excess levels of galactose receptor on membranes varied (46.5%, 40.2%, and 4.7% for CEA-1, -2, and -3, respectively). These CEAs were shown to be 2.3%, 7.9%, and 0.7% as effective, respectively, as asialo-alpha 1-acid glycoprotein in inhibiting the binding of radiolabeled asialo-alpha 1-acid glycoprotein to liver cell membranes. Each of the three CEA preparations showed different clearance kinetics from the circulation of mice. Coinjection of asialo-alpha 1-acid glycoprotein with the CEAs revealed differing inhibition of the clearances. These results show that differences in the carbohydrate components of purified CEA preparations affect their rate of removal from circulation and thus possibly the relationship between CEA production and observed plasma levels in patients. The possible origin of these CEA differences is discussed with their clinical implications.

NMR binding and crystal structure reveal that intrinsically-unstructured regulatory domain auto-inhibits PAK4 by a mechanism different from that of PAK1.

PubMed

Wang, Wei; Lim, Liangzhong; Baskaran, Yohendran; Manser, Ed; Song, Jianxing

2013-08-16

Six human PAK members are classified into groups I (PAKs 1-3) and II (PAK4-6). Previously, only group I PAKs were thought to be auto-inhibited but very recently PAK4, the prototype of group II PAKs, has also been shown to be auto-inhibited by its N-terminal regulatory domain. However, the complete auto-inhibitory domain (AID) sequence remains undefined and the mechanism underlying its auto-inhibition is largely elusive. Here, the N-terminal regulatory domain of PAK4 sufficient for auto-inhibiting and binding Cdc42/Rac was characterized to be intrinsically unstructured, but nevertheless we identified the entire AID sequence by NMR. Strikingly, an AID peptide was derived by deleting the binding-unnecessary residues, which has a Kd of 320 nM to the PAK4 catalytic domain. Consequently, the PAK4 crystal structure complexed with the entire AID has been determined, which reveals that the complete kinase cleft is occupied by 20 AID residuescomposed of an N-terminal α-helix and a previously-identified pseudosubstrate motif, thus achieving auto-inhibition. Our study reveals that PAK4 is auto-inhibited by a novel mechanism which is completely different from that for PAK1, thus bearing critical implications for design of inhibitors specific for group II PAKs. Copyright © 2013 Elsevier Inc. All rights reserved.

Characterization of strychnine-sensitive glycine receptor in the intact frog retina: modulation by protein kinases.

PubMed

Salceda, Rocío; Aguirre-Ramirez, Marisela

2005-03-01

We studied 3H-glycine and 3H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a KD of 2.5 and 2.0 microM, respectively. Specific binding of glycine was displaced by beta-alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

PubMed

Giuntini, Serena; Beernink, Peter T; Reason, Donald C; Granoff, Dan M

2012-01-01

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

Consequences of ChemR23 Heteromerization with the Chemokine Receptors CXCR4 and CCR7

PubMed Central

de Poorter, Cédric; Baertsoen, Kevin; Lannoy, Vincent; Parmentier, Marc; Springael, Jean-Yves

2013-01-01

Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed. PMID:23469143

Consequences of ChemR23 heteromerization with the chemokine receptors CXCR4 and CCR7.

PubMed

de Poorter, Cédric; Baertsoen, Kevin; Lannoy, Vincent; Parmentier, Marc; Springael, Jean-Yves

2013-01-01

Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.

High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

PubMed

Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

2014-01-17

The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

Selective labeling of serotonin uptake sites in rat brain by (/sup 3/H)citalopram contrasted to labeling of multiple sites by (/sup 3/H)imipramine

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Amato, R.J.; Largent, B.L.; Snowman, A.M.

1987-07-01

Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes (/sup 3/H)citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for (/sup 3/H)citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of (/sup 3/H)citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of (/sup 3/H)citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of (/sup 3/H)imipramine bindingmore » reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific (/sup 3/H)imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of (/sup 3/H)imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in (/sup 3/H)citalopram and serotonin-sensitive (/sup 3/H)imipramine binding with only a small effect on serotonin-insensitive (/sup 3/H)imipramine binding. The dissociation rate of (/sup 3/H)imipramine or (/sup 3/H)citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive (/sup 3/H)imipramine high affinity binding sites closely resembles that of (/sup 3/H)citalopram binding.« less

Assessing Specific Oligonucleotides and Small Molecule Antibiotics for the Ability to Inhibit the CRD-BP-CD44 RNA Interaction

PubMed Central

Thomsen, Dana; Lee, Chow H.

2014-01-01

Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions. PMID:24622399

Assessing specific oligonucleotides and small molecule antibiotics for the ability to inhibit the CRD-BP-CD44 RNA interaction.

PubMed

King, Dustin T; Barnes, Mark; Thomsen, Dana; Lee, Chow H

2014-01-01

Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3'UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862-3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862-3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions.

Inhibition of pneumococcal choline-binding proteins and cell growth by esters of bicyclic amines.

PubMed

Maestro, Beatriz; González, Ana; García, Pedro; Sanz, Jesús M

2007-01-01

Streptococcus pneumoniae is one of the major pathogens worldwide. The use of currently available antibiotics to treat pneumococcal diseases is hampered by increasing resistance levels; also, capsular polysaccharide-based vaccination is of limited efficacy. Therefore, it is desirable to find targets for the development of new antimicrobial drugs specifically designed to fight pneumococcal infections. Choline-binding proteins are a family of polypeptides, found in all S. pneumoniae strains, that take part in important physiologic processes of this bacterium. Among them are several murein hydrolases whose enzymatic activity is usually inhibited by an excess of choline. Using a simple chromatographic procedure, we have identified several choline analogs able to strongly interact with the choline-binding module (C-LytA) of the major autolysin of S. pneumoniae. Two of these compounds (atropine and ipratropium) display a higher binding affinity to C-LytA than choline, and also increase the stability of the protein. CD and fluorescence spectroscopy analyses revealed that the conformational changes of C-LytA upon binding of these alkaloids are different to those induced by choline, suggesting a different mode of binding. In vitro inhibition assays of three pneumococcal, choline-dependent cell wall lytic enzymes also demonstrated a greater inhibitory efficiency of those molecules. Moreover, atropine and ipratropium strongly inhibited in vitro pneumococcal growth, altering cell morphology and reducing cell viability, a very different response than that observed upon addition of an excess of choline. These results may open up the possibility of the development of bicyclic amines as new antimicrobials for use against pneumococcal pathologies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Honda, T.; Adachi, H.; Noguchi, M.

The authors have examined the effect of carbamylcholine on the binding of cholecystokinin (CCK) to dispersed acini from rat pancreas. The CCK receptor on pancreatic acini possesses two classes of binding sites. Simultaneous addition of carbamylcholine inhibited binding of CCK binding sites. Atropine prevented the inhibitory effect of carbamylcholine, whereas calcium ionophore A23187 did not alter binding of CCK. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited binding of CCK in the same manner as carbamylcholine. Inhibition by carbamylcholine was reversible and the recovery was time dependent. By contrast, inhibition of binding of CCK by TPA did not reverse after a 60-min incubation without themore » agent. These findings, at least in part, account for the inhibition of the CCK-induced stimulation of amylase secretion by carbamylcholine. The action of TPA on binding of CCK suggests the possible involvement of the activation of protein kinase C in the inhibition of binding.« less

Increased expression of Aspergillus parasiticus aflR, encoding a sequence-specific DNA-binding protein, relieves nitrate inhibition of aflatoxin biosynthesis.

PubMed Central

Chang, P K; Ehrlich, K C; Yu, J; Bhatnagar, D; Cleveland, T E

1995-01-01

The aflR gene from Aspergillus parasiticus and Aspergillus flavus may be involved in the regulation of aflatoxin biosynthesis. The aflR gene product, AFLR, possesses a GAL4-type binuclear zinc finger DNA-binding domain. A transformant, SU1-N3 (pHSP), containing an additional copy of aflR, showed increased transcription of aflR and the aflatoxin pathway structural genes, nor-1, ver-1, and omt-1, when cells were grown in nitrate medium, which normally suppresses aflatoxin production. Electrophoretic mobility shift assays showed that the recombinant protein containing the DNA-binding domain, AFLR1, bound specifically to the palindromic sequence, TTAGGCCTAA, 120 bp upstream of the AFLR translation start site. Expression of aflR thus appears to be autoregulated. Increased expression of aflatoxin biosynthetic genes in the transformant might result from an elevated basal level of AFLR, allowing it to overcome nitrate inhibition and to bind to the aflR promotor region, thereby initiating aflatoxin biosynthesis. Results further suggest that aflR is involved in the regulation of multiple parts of the aflatoxin biosynthetic pathway. PMID:7793958

Antiadhesive Properties of Abelmoschus esculentus (Okra) Immature Fruit Extract against Helicobacter pylori Adhesion

PubMed Central

Shevtsova, Anna; Glocker, Erik; Borén, Thomas; Hensel, Andreas

2014-01-01

Background Traditional Asian and African medicine use immature okra fruits (Abelmoschus esculentus) as mucilaginous food to combat gastritis. Its effectiveness is due to polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach tissue. The present study investigates the antiadhesive effect in mechanistic detail. Methodology A standardized aqueous fresh extract (Okra FE) from immature okra fruits was used for a quantitative in vitro adhesion assay with FITC-labled H. pylori J99, 2 clinical isolates, AGS cells, and fluorescence-activated cell sorting. Bacterial adhesins affected by FE were pinpointed using a dot-blot overlay assay with immobilized Lewisb, sialyl-Lewisa, H-1, laminin, and fibronectin. 125I-radiolabeled Okra FE polymer served for binding studies to different H. pylori strains and interaction experiments with BabA and SabA. Iron nanoparticles with different coatings were used to investigate the influence of the charge-dependence of an interaction on the H. pylori surface. Principal findings Okra FE dose-dependently (0.2 to 2 mg/mL) inhibited H. pylori binding to AGS cells. FE inhibited the adhesive binding of membrane proteins BabA, SabA, and HpA to its specific ligands. Radiolabeled compounds from FE bound non-specifically to different strains of H. pylori, as well as to BabA/SabA deficient mutants, indicating an interaction with a still-unknown membrane structure in the vicinity of the adhesins. The binding depended on the charge of the inhibitors. Okra FE did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors. Conclusion Non-specific interactions between high molecular compounds from okra fruits and the H. pylori surface lead to strong antiadhesive effects. PMID:24416297

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bidart, J.M.; Troalen, F.; Salesse, R.

1987-06-25

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptidesmore » spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.« less

Synergistic regulation of competence development in Bacillus subtilis by two Rap-Phr systems.

PubMed

Bongiorni, Cristina; Ishikawa, Shu; Stephenson, Sophie; Ogasawara, Naotake; Perego, Marta

2005-07-01

The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is involved in the regulation of competence to DNA transformation. The ComA response regulator and transcription factor for initiation of competence development is the target of RapF. Specific binding of RapF to the carboxy-terminal DNA-binding domain of ComA inhibits the response regulator's ability to bind its target DNA promoters. The PhrF C-terminal pentapeptide, QRGMI, inhibits RapF activity. The activity of RapF and PhrF in regulating competence development is analogous to the previously described activity of RapC and PhrC (L. J. Core and M. Perego, Mol. Microbiol. 49:1509-1522, 2003). In fact, the RapF and PhrF pair of proteins acts synergistically with RapC and PhrC in the overall regulation of the ComA transcription factor. Since the transcription of the RapC- and RapF-encoding genes is positively regulated by their own target ComA, an autoregulatory circuit must exist for the competence transcription factor in order to modulate its activity.

Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A.

PubMed

Grover, Abhinav; Agrawal, Vibhuti; Shandilya, Ashutosh; Bisaria, Virendra S; Sundar, Durai

2011-01-01

Herpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug. Withaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured. We have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies will also guide the design of selective inhibitors of DNA POL with high specificity and potent activity in order to strengthen the therapeutic arsenal available today against the dangerous biological warfare agent represented by Herpes Simplex Virus.

Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A

PubMed Central

2011-01-01

Background Herpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug. Results Withaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured. Conclusions We have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies will also guide the design of selective inhibitors of DNA POL with high specificity and potent activity in order to strengthen the therapeutic arsenal available today against the dangerous biological warfare agent represented by Herpes Simplex Virus. PMID:22373101

Herpes simplex virus type 1 tegument protein VP22 interacts with TAF-I proteins and inhibits nucleosome assembly but not regulation of histone acetylation by INHAT.

PubMed

van Leeuwen, Hans; Okuwaki, Mitsuru; Hong, Rui; Chakravarti, Debabrata; Nagata, Kyosuke; O'Hare, Peter

2003-09-01

Affinity chromatography was used to identify cellular proteins that interact with the herpes simplex virus (HSV) tegument protein VP22. Among a small set of proteins that bind specifically to VP22, we identified TAF-I (template-activating factor I), a chromatin remodelling protein and close homologue of the histone chaperone protein NAP-1. TAF-I has been shown previously to promote more ordered transfer of histones to naked DNA through a direct interaction with histones. TAF-I, as a subunit of the INHAT (inhibitor of acetyltransferases) protein complex, also binds to histones and masks them from being substrates for the acetyltransferases p300 and PCAF. Using in vitro assays for TAF-I activity in chromatin assembly, we show that VP22 inhibits nucleosome deposition on DNA by binding to TAF-I. We also observed that VP22 binds non-specifically to DNA, an activity that is abolished by TAF-I. However, the presence of VP22 does not affect the property of INHAT in inhibiting the histone acetyltransferase activity of p300 or PCAF in vitro. We speculate that this interaction could be relevant to HSV DNA organization early in infection, for example, by interfering with nucleosomal deposition on the genome. Consistent with this possibility was the observation that overexpression of TAF-I in transfected cells interferes with the progression of HSV-1 infection.

Characterization of the slow calcium channel binding sites for ( sup 3 H)SR 33557 in rat heart sarcolemmal membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chatelain, P.; Beaufort, P.; Meysmans, L.

1991-01-01

SR 33557 represents a new class of compounds (indolizine sulfone) that inhibit L-type Ca2+ channels. ({sup 3}H)SR 33557 has been shown to bind with high affinity (Kd congruent to 0.36 nM, calculated from saturation isotherms and association/dissociation kinetics) to a single class of sites in a purified preparation of rat cardiac sarcolemmal membranes. The binding was found to be saturable and reversible. The maximal binding capacity was in approximately 1:1 stoichiometry with that of other Ca2+ channel antagonists. Various divalent cations (Mg2+, Mn2+, Ca2+, Ba2+, and Cd2+) were shown to inhibit specific ({sup 3}H)SR 33557 binding, with Cd2+ being themore » most potent. Among several receptor or channel ligands (including omega-conotoxin and Na+ and K+ channel modulators), only the L-type Ca2+ channel antagonists were found to displace ({sup 3}H)SR 33557. However, dihydropyridines, phenylalkylamines, benzothiazepines, and diphenylbutylpiperidines were found to inhibit ({sup 3}H)SR 33557 in a noncompetitive manner as demonstrated by displacement and saturation experiments in addition to dissociation kinetics. From these results, we suggest that SR 33557 binds with high affinity to a unique site on the L-type Ca2+ channel found in rat cardiac sarcolemmal membranes.« less

Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide.

PubMed

Diaz, Alejandra R; Core, Leighton J; Jiang, Min; Morelli, Michela; Chiang, Christina H; Szurmant, Hendrik; Perego, Marta

2012-03-01

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.

Bacillus subtilis RapA Phosphatase Domain Interaction with Its Substrate, Phosphorylated Spo0F, and Its Inhibitor, the PhrA Peptide

PubMed Central

Diaz, Alejandra R.; Core, Leighton J.; Jiang, Min; Morelli, Michela; Chiang, Christina H.; Szurmant, Hendrik

2012-01-01

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms. PMID:22267516

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin

PubMed Central

Sato, Yuichiro; Morimoto, Kinjiro; Kubo, Takanori; Sakaguchi, Takemasa; Nishizono, Akira; Hirayama, Makoto; Hori, Kanji

2015-01-01

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC50 of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC50s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells. PMID:26035023

Functional somatostatin receptors on a rat pancreatic acinar cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.

1988-07-01

Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibitionmore » of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.« less

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3)

PubMed Central

Koymans, Kirsten J.; Feitsma, Louris J.; Brondijk, T. Harma C.; Aerts, Piet C.; Lukkien, Eddie; Lössl, Philip; van Kessel, Kok P. M.; de Haas, Carla J. C.; van Strijp, Jos A. G.; Huizinga, Eric G.

2015-01-01

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2–glycans with the conserved LewisX binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2–Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2–lipopeptide complex, it prevents TLR heterodimerization and downstream signaling. PMID:26283364

Silibinin inhibits aberrant lipid metabolism, proliferation and emergence of androgen-independence in prostate cancer cells via primarily targeting the sterol response element binding protein 1

PubMed Central

Nambiar, Dhanya K.; Deep, Gagan; Singh, Rana P.; Agarwal, Chapla; Agarwal, Rajesh

2014-01-01

Prostate cancer (PCA) kills thousands of men every year, demanding additional approaches to better understand and target this malignancy. Recently, critical role of aberrant lipogenesis is highlighted in prostate carcinogenesis, offering a unique opportunity to target it to reduce PCA. Here, we evaluated efficacy and associated mechanisms of silibinin in inhibiting lipid metabolism in PCA cells. At physiologically achievable levels in human, silibinin strongly reduced lipid and cholesterol accumulation specifically in human PCA cells but not in non-neoplastic prostate epithelial PWR-1E cells. Silibinin also decreased nuclear protein levels of sterol regulatory element binding protein 1 and 2 (SREBP1/2) and their target genes only in PCA cells. Mechanistically, silibinin activated AMPK, thereby increasing SREBP1 phosphorylation and inhibiting its nuclear translocation; AMPK inhibition reversed silibinin-mediated decrease in nuclear SREBP1 and lipid accumulation. Additionally, specific SREBP inhibitor fatostatin and stable overexpression of SREBP1 further confirmed the central role of SREBP1 in silibinin-mediated inhibition of PCA cell proliferation and lipid accumulation and cell cycle arrest. Importantly, silibinin also inhibited synthetic androgen R1881-induced lipid accumulation and completely abrogated the development of androgen-independent LNCaP cell clones via targeting SREBP1/2. Together, these mechanistic studies suggest that silibinin would be effective against PCA by targeting critical aberrant lipogenesis. PMID:25294820

Silibinin inhibits aberrant lipid metabolism, proliferation and emergence of androgen-independence in prostate cancer cells via primarily targeting the sterol response element binding protein 1.

PubMed

Nambiar, Dhanya K; Deep, Gagan; Singh, Rana P; Agarwal, Chapla; Agarwal, Rajesh

2014-10-30

Prostate cancer (PCA) kills thousands of men every year, demanding additional approaches to better understand and target this malignancy. Recently, critical role of aberrant lipogenesis is highlighted in prostate carcinogenesis, offering a unique opportunity to target it to reduce PCA. Here, we evaluated efficacy and associated mechanisms of silibinin in inhibiting lipid metabolism in PCA cells. At physiologically achievable levels in human, silibinin strongly reduced lipid and cholesterol accumulation specifically in human PCA cells but not in non-neoplastic prostate epithelial PWR-1E cells. Silibinin also decreased nuclear protein levels of sterol regulatory element binding protein 1 and 2 (SREBP1/2) and their target genes only in PCA cells. Mechanistically, silibinin activated AMPK, thereby increasing SREBP1 phosphorylation and inhibiting its nuclear translocation; AMPK inhibition reversed silibinin-mediated decrease in nuclear SREBP1 and lipid accumulation. Additionally, specific SREBP inhibitor fatostatin and stable overexpression of SREBP1 further confirmed the central role of SREBP1 in silibinin-mediated inhibition of PCA cell proliferation and lipid accumulation and cell cycle arrest. Importantly, silibinin also inhibited synthetic androgen R1881-induced lipid accumulation and completely abrogated the development of androgen-independent LNCaP cell clones via targeting SREBP1/2. Together, these mechanistic studies suggest that silibinin would be effective against PCA by targeting critical aberrant lipogenesis.

Sex-lethal promotes nuclear retention of msl2 mRNA via interactions with the STAR protein HOW

PubMed Central

Graindorge, Antoine; Carré, Clément; Gebauer, Fátima

2013-01-01

Female-specific repression of male-specific-lethal-2 (msl2) mRNA in Drosophila melanogaster provides a paradigm for coordinated control of gene expression by RNA-binding complexes. Repression is orchestrated by Sex-lethal (SXL), which binds to the 5′ and 3′ untranslated regions (UTRs) of the mRNA and inhibits splicing in the nucleus and subsequent translation in the cytoplasm. Here we show that SXL ensures msl2 silencing by yet a third mechanism that involves inhibition of nucleocytoplasmic transport of msl2 mRNA. To identify SXL cofactors in msl2 regulation, we devised a two-step purification method termed GRAB (GST pull-down and RNA affinity binding) and identified Held-Out-Wings (HOW) as a component of the msl2 5′ UTR-associated complex. HOW directly interacts with SXL and binds to two sequence elements in the msl2 5′ UTR. Depletion of HOW reduces the capacity of SXL to repress the expression of msl2 reporters without affecting SXL-mediated regulation of splicing or translation. Instead, HOW is required for SXL to retain msl2 transcripts in the nucleus. Cooperation with SXL confers a sex-specific role to HOW. Our results uncover a novel function of SXL in nuclear mRNA retention and identify HOW as a mediator of this function. PMID:23788626

Bacterial Flagellin-Specific Chaperone FliS Interacts with Anti-Sigma Factor FlgM

PubMed Central

Galeva, Anna; Moroz, Natalia; Yoon, Young-Ho; Hughes, Kelly T.; Samatey, Fadel A.

2014-01-01

Flagella are extracellular organelles that propel bacteria. Each flagellum consists of a basal body, a hook, and a filament. The major protein of the filament is flagellin. Induction of flagellin gene expression coincides with secretion of FlgM. The role of FlgM is to inhibit FliA (σ28), a flagellum-specific RNA polymerase responsible for flagellin transcription. To prevent premature polymerization of newly synthesized flagellin molecules, FliS, the flagellin-specific chaperone, binds flagellin and facilitates its export. In this study, the interaction between FlgM and FliS from Salmonella enterica serovar Typhimurium was characterized using gel shift, intrinsic tryptophan fluorescence, circular dichroism, limited proteolysis, and cross-linking. We have demonstrated that (i) FliS and FlgM interact specifically, forming a 1:1 complex, (ii) the FliS binding site on FlgM is proximal to or even overlaps the binding site for FliA, and (iii) FliA competes with FliS for FlgM binding. PMID:24415724

A Sequence in the loop domain of hepatitis C virus E2 protein identified in silico as crucial for the selective binding to human CD81

PubMed Central

Chang, Chun-Chun; Hsu, Hao-Jen; Yen, Jui-Hung; Lo, Shih-Yen

2017-01-01

Hepatitis C virus (HCV) is a species-specific pathogenic virus that infects only humans and chimpanzees. Previous studies have indicated that interactions between the HCV E2 protein and CD81 on host cells are required for HCV infection. To determine the crucial factors for species-specific interactions at the molecular level, this study employed in silico molecular docking involving molecular dynamic simulations of the binding of HCV E2 onto human and rat CD81s. In vitro experiments including surface plasmon resonance measurements and cellular binding assays were applied for simple validations of the in silico results. The in silico studies identified two binding regions on the HCV E2 loop domain, namely E2-site1 and E2-site2, as being crucial for the interactions with CD81s, with the E2-site2 as the determinant factor for human-specific binding. Free energy calculations indicated that the E2/CD81 binding process might follow a two-step model involving (i) the electrostatic interaction-driven initial binding of human-specific E2-site2, followed by (ii) changes in the E2 orientation to facilitate the hydrophobic and van der Waals interaction-driven binding of E2-site1. The sequence of the human-specific, stronger-binding E2-site2 could serve as a candidate template for the future development of HCV-inhibiting peptide drugs. PMID:28481946

Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates

PubMed Central

1977-01-01

A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D- galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies. PMID:833547

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niu, Mingshan; Jiangsu Key Laboratory of Bone Marrow Stem Cell, Xuzhou Medical College, Xuzhou, Jiangsu; Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu

Constitutive NF-κB activation is required for survival of activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL). However, current NF-κB targeting strategies lack cancer cell specificity. Here, we identified a novel inhibitor, piperlongumine, features direct binding to NF-κB p65 subunit and suppression of p65 nuclear import. This was accompanied by NF-κB reporter activity suppression and NF-κB target gene downregulation. Moreover, mutation of Cys{sup 38} to Ser in p65 abolished this effect of piperlongumine on inhibition of p65 nuclear import. Furthermore, we show that piperlongumine selectively inhibited proliferation and induced apoptosis of ABC-DLBCL cells. Most notably, it has beenmore » reported that piperlongumine did not affect normal cells even at high doses and was nontoxic to animals. Hence, our current study provides new insight into piperlongumine's mechanism of action and novel approach to ABC-DLBCL target therapy. - Highlights: • Current NF-κB targeting strategies lack cancer cell specificity. • Piperlongumine inhibits NF-κB p65 subunit nuclear import via directly binding to p65. • Piperlongumine selectively inhibits proliferation of ABC-DLBCL cells. • This study provides a novel approach to ABC-DLBCL target therapy.« less

Novel dengue virus NS2B/NS3 protease inhibitors.

PubMed

Wu, Hongmei; Bock, Stefanie; Snitko, Mariya; Berger, Thilo; Weidner, Thomas; Holloway, Steven; Kanitz, Manuel; Diederich, Wibke E; Steuber, Holger; Walter, Christof; Hofmann, Daniela; Weißbrich, Benedikt; Spannaus, Ralf; Acosta, Eliana G; Bartenschlager, Ralf; Engels, Bernd; Schirmeister, Tanja; Bodem, Jochen

2015-02-01

Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Development of a Fluorescence Assay for the Characterization of Brevenal Binding to Rat Brain Synaptosomes

PubMed Central

2015-01-01

The marine dinoflagellate Karenia brevis produces a family of neurotoxins known as brevetoxins. Brevetoxins elicit their effects by binding to and activating voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces brevenal, a brevetoxin antagonist, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. In this study, we have developed a novel fluorescence binding assay for the brevenal binding site. Several fluorescent compounds were conjugated to brevenal to assess their effects on brevenal binding. The assay was validated against the radioligand assay for the brevenal binding site and yielded comparable equilibrium inhibition constants. The fluorescence-based assay was shown to be quicker and far less expensive and did not generate radioactive waste or need facilities for handling radioactive materials. In-depth studies using the brevenal conjugates showed that, while brevenal conjugates do bind to a binding site in the VSSC protein complex, they are not displaced by known VSSC site specific ligands. As such, brevenal elicits its action through a novel mechanism and/or currently unknown receptor site on VSSCs. PMID:25226846

Specific electrostatic interactions between charged amino acid residues regulate binding of von Willebrand factor to blood platelets.

PubMed

Interlandi, Gianluca; Yakovenko, Olga; Tu, An-Yue; Harris, Jeff; Le, Jennie; Chen, Junmei; López, José A; Thomas, Wendy E

2017-11-10

The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet-binding A1 domain of VWF is connected to the VWF N-terminally located D'D3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ibα (GpIbα). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated. Here, we show that an aspartate at position 1261 is the most critical residue of the N-terminal linker for inhibiting binding of the VWF A1 domain to GpIbα on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1-GpIbα binding experiments, we identified a network of salt bridges between Asp 1261 and the rest of A1 that lock the N-terminal linker in place such that it reduces binding to GpIbα. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIbα, in which case the mutations inhibited the binding. These results show that interactions between charged amino acid residues are important both to directly stabilize the A1-GpIbα complex and to indirectly destabilize the complex through the N-terminal linker. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs

PubMed Central

Scott, Fiona L; Denault, Jean-Bernard; Riedl, Stefan J; Shin, Hwain; Renatus, Martin; Salvesen, Guy S

2005-01-01

The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP. PMID:15650747

Perinatal MAO Inhibition Produces Long-Lasting Impairment of Serotonin Function in Offspring.

PubMed

Burke, Mark W; Fillion, Myriam; Mejia, Jose; Ervin, Frank R; Palmour, Roberta M

2018-06-11

In addition to transmitter functions, many neuroamines have trophic or ontogenetic regulatory effects important to both normal and disordered brain development. In previous work (Mejia et al., 2002), we showed that pharmacologically inhibiting monoamine oxidase (MAO) activity during murine gestation increases the prevalence of behaviors thought to reflect impulsivity and aggression. The goal of the present study was to determine the extent to which this treatment influences dopamine and serotonin innervation of murine cortical and subcortical areas, as measured by regional density of dopamine (DAT) and serotonin transporters (SERT). We measured DAT and SERT densities at 3 developmental times (PND 14, 35 and 90) following inhibition of MAO A, or MAO B or both throughout murine gestation and early post-natal development. DAT binding was unaltered within the nigrostriatal pathway, but concurrent inhibition of MAO-A and MAO-B significantly and specifically reduced SERT binding by 10⁻25% in both the frontal cortex and raphe nuclei. Low levels of SERT binding persisted (PND 35, 90) after the termination (PND 21) of exposure to MAO inhibitors and was most marked in brain structures germane to the previously described behavioral changes. The relatively modest level of enzyme inhibition (25⁻40%) required to produce these effects mandates care in the use of any compound which might inhibit MAO activity during gestation.

Functional diversification of ROK-family transcriptional regulators of sugar catabolism in the Thermotogae phylum

PubMed Central

Kazanov, Marat D.; Li, Xiaoqing; Gelfand, Mikhail S.; Osterman, Andrei L.; Rodionov, Dmitry A.

2013-01-01

Large and functionally heterogeneous families of transcription factors have complex evolutionary histories. What shapes specificities toward effectors and DNA sites in paralogous regulators is a fundamental question in biology. Bacteria from the deep-branching lineage Thermotogae possess multiple paralogs of the repressor, open reading frame, kinase (ROK) family regulators that are characterized by carbohydrate-sensing domains shared with sugar kinases. We applied an integrated genomic approach to study functions and specificities of regulators from this family. A comparative analysis of 11 Thermotogae genomes revealed novel mechanisms of transcriptional regulation of the sugar utilization networks, DNA-binding motifs and specific functions. Reconstructed regulons for seven groups of ROK regulators were validated by DNA-binding assays using purified recombinant proteins from the model bacterium Thermotoga maritima. All tested regulators demonstrated specific binding to their predicted cognate DNA sites, and this binding was inhibited by specific effectors, mono- or disaccharides from their respective sugar catabolic pathways. By comparing ligand-binding domains of regulators with structurally characterized kinases from the ROK family, we elucidated signature amino acid residues determining sugar-ligand regulator specificity. Observed correlations between signature residues and the sugar-ligand specificities provide the framework for structure functional classification of the entire ROK family. PMID:23209028

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

PubMed

Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

1992-08-05

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Mimotopes for Api g 5, a Relevant Cross-reactive Allergen, in the Celery-Mugwort-Birch-Spice Syndrome.

PubMed

Lukschal, Anna; Wallmann, Julia; Bublin, Merima; Hofstetter, Gerlinde; Mothes-Luksch, Nadine; Breiteneder, Heimo; Pali-Schöll, Isabella; Jensen-Jarolim, Erika

2016-03-01

In the celery-mugwort-birch-spice syndrome, a significant proportion of IgE is directed against high molecular weight (HMW) glycoproteins, including the celery allergen Api g 5. BIP3, a monoclonal antibody originally raised against birch pollen, recognizes HMW allergens in birch and mugwort pollens, celery, and Apiaceae spices. Our aim was to generate mimotopes using BIP3 for immunization against the HMW allergens relevant in the celery-mugwort-birch-spice cross reactivity syndrome. Mimotopes were selected from a random-peptide display library by BIP3 and applied in IgE inhibition assays. The 3 phage clones with the highest inhibitory capacity were chosen for immunization of BALB/c mice. Mouse immune sera were tested for IgG binding to blotted birch pollen extract and used for inhibiting patients' IgE binding. Furthermore, sera were tested for binding to Api g 5, to horseradish peroxidase (HRP) as a second glycoprotein, or to non-glycosylated control allergen Phl p 5 in ELISA, and the specific Api g 5-specific IgG titers were determined. Three rounds of biopanning resulted in phage clones exhibiting 7 different sequences including 1 dominant, 1-6-cyclo-CHKLRCDKAIA. Three phage clones had the capacity to inhibit human IgE binding and induced IgG to the HMW antigen when used for immunizing BALB/c mice. The induced BIP3-mimotope IgG reached titers of 1:500 specifically to Api g 5, but hardly reacted to glycoprotein HRP, revealing a minor role of carbohydrates in their epitope. The mimotopes characterized in this study mimic the epitope of BIP3 relevant for Api g 5, one of the cross-reactive HMW allergens relevant in the celery-mugwort-birch-spice syndrome. BIP3 mimotopes may be used in the future for hyposensitization in this clinical syndrome by virtue of good and specific immunogenicity.

Cyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor.

PubMed

Nkongolo, Shirin; Ni, Yi; Lempp, Florian A; Kaufman, Christina; Lindner, Thomas; Esser-Nobis, Katharina; Lohmann, Volker; Mier, Walter; Mehrle, Stefan; Urban, Stephan

2014-04-01

Chronic hepatitis B and hepatitis D are global health problems caused by the human hepatitis B and hepatitis D virus. The myristoylated preS1 domain of the large envelope protein mediates specific binding to hepatocytes by sodium taurocholate co-transporting polypeptide (NTCP). NTCP is a bile salt transporter known to be inhibited by cyclosporin A. This study aimed to characterize the effect of cyclosporin A on HBV/HDV infection. HepaRG cells, primary human hepatocytes, and susceptible NTCP-expressing hepatoma cell lines were applied for infection experiments. The mode of action of cyclosporin A was studied by comparing the effect of different inhibitors, cyclophilin A/B/C-silenced cell lines as well as NTCP variants and mutants. Bile salt transporter and HBV receptor functions were investigated by taurocholate uptake and quantification of HBVpreS binding. Cyclosporin A inhibited hepatitis B and D virus infections during and--less pronounced--prior to virus inoculation. Binding of HBVpreS to NTCP was blocked by cyclosporin A concentrations at 8 μM. An NTCP variant deficient in HBVpreS binding but competent for bile salt transport showed resistance to cyclosporin A. Silencing of cyclophilins A/B/C did not abrogate transporter and receptor inhibition. In contrast, tacrolimus, a cyclophilin-independent calcineurin inhibitor, was inactive. HBV and HDV entry via sodium taurocholate co-transporting polypeptide is inhibited by cyclosporin A. The interaction between the drug and the viral receptor is direct and overlaps with a functional binding site of the preS1 domain, which mediates viral entry. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

C-reactive protein specifically binds to Fcgamma receptor type I on a macrophage-like cell line.

PubMed

Tron, Kyrylo; Manolov, Dimitar E; Röcker, Carlheinz; Kächele, Martin; Torzewski, Jan; Nienhaus, G Ulrich

2008-05-01

C-reactive protein (CRP) is a prototype acute-phase protein that may be intimately involved in human disease. Its cellular receptors are still under debate; the main candidates are FcR for immunoglobulin G, as CRP was shown to bind specifically to FcgammaRI and FcgammaRIIa. Using ultrasensitive confocal live-cell imaging, we have studied CRP binding to FcgammaR naturally expressed in the plasma membranes of cells from a human leukemia cell line (Mono Mac 6). These macrophage-like cells express high levels of FcgammaRI and FcgammaRII. They were shown to bind fluorescently labeled CRP with micromolar affinity, KD = (6.6 +/- 1.5) microM. CRP binding could be inhibited by pre-incubation with human but not mouse IgG and was thus FcgammaR-specific. Blocking of FcgammaRI by an FcgammaRI-specific antibody abolished CRP binding essentially completely, whereas application of antibodies against FcgammaRII did not have a noticeable effect. In fluorescence images of Mono Mac 6 cells, the intensity patterns of bound CRP were correlated with those of FcgammaRI, but not FcgammaRII. These results provide clear evidence of specific interactions between CRP and FcgammaR (predominantly FcgammaRI) naturally expressed on macrophage-like cells.

New Insights into the Mechanism of Inhibition of p53 by Simian Virus 40 Large T Antigen

PubMed Central

Sheppard, Hilary M.; Corneillie, Siska I.; Espiritu, Christine; Gatti, Andrea; Liu, Xuan

1999-01-01

Simian virus 40 (SV40) large tumor antigen (T antigen) has been shown to inhibit p53-dependent transcription by preventing p53 from binding to its cognate cis element. Data presented in this report provide the first direct functional evidence that T antigen, under certain conditions, may also repress p53-dependent transcription by a mechanism in which the transactivation domain of p53 is abrogated while DNA binding is unaffected. Specifically, p53 purified as a complex with T antigen from mouse cells was found to bind DNA as a transcriptionally inactive intact complex, while that purified from human cells was found to bind DNA independently of T antigen and could activate p53-dependent transcription. This difference in activity may be dependent on a different interaction of T antigen with mouse and human p53 and, in addition, on the presence of super T, which is found only in transformed rodent cells. These results suggest that subtle yet important differences exist between the inhibition of p53 by T antigen in mouse and human cells. The implications of this finding with respect to SV40-associated malignancies are discussed. PMID:10082540

A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC

PubMed Central

Kenesi, Erzsébet; Lózsa, Rita

2017-01-01

Abstract In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine. PMID:28499009

An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

NASA Astrophysics Data System (ADS)

Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

1998-04-01

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus

PubMed Central

Gao, Xiang; Esseili, Malak A.; Lu, Zhongyan; Saif, Linda J.

2016-01-01

ABSTRACT Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. IMPORTANCE Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination. PMID:26969699

Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus.

PubMed

Gao, Xiang; Esseili, Malak A; Lu, Zhongyan; Saif, Linda J; Wang, Qiuhong

2016-05-15

Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Carbohydrate binding properties of the stinging nettle (Urtica dioica) rhizome lectin.

PubMed

Shibuya, N; Goldstein, I J; Shafer, J A; Peumans, W J; Broekaert, W F

1986-08-15

The interaction of the stinging nettle rhizome lectin (UDA) with carbohydrates was studied by using the techniques of quantitative precipitation, hapten inhibition, equilibrium dialysis, and uv difference spectroscopy. The Carbohydrate binding site of UDA was determined to be complementary to an N,N',N"-triacetylchitotriose unit and proposed to consist of three subsites, each of which has a slightly different binding specificity. UDA also has a hydrophobic interacting region adjacent to the carbohydrate binding site. Equilibrium dialysis and uv difference spectroscopy revealed that UDA has two carbohydrate binding sites per molecule consisting of a single polypeptide chain. These binding sites either have intrinsically different affinities for ligand molecules, or they may display negative cooperativity toward ligand binding.

Compound-Specific Effects of Mutations at Val787 in DII-S6 of Nav1.4 Sodium Channels on the Action of Sodium Channel Inhibitor Insecticides

PubMed Central

von Stein, Richard T.; Soderlund, David M.

2012-01-01

Sodium channel inhibitor (SCI) insecticides are hypothesized to inhibit voltage-gated sodium channels by binding selectively to the slow-inactivated state. Replacement of valine at position 787 in the S6 segment of homology domain II of the rat Nav1.4 sodium channel by lysine (V787K) enchances slow inactivation of this channel whereas replacement by alanine or cysteine (V787A, V787C) inhibits slow inactivation. To test the hypothesis that SCI insecticides bind selectively to the slow-inactivated state, we constructed mutated Nav1.4/V787A, Nav1.4/V787C, and Nav1.4/V787K cDNAs, expressed wildtype and mutated channels with the auxiliary β1 subunit in Xenopus oocytes, and used the two-electrode voltage clamp technique to examine the effects of these mutations on channel inhibition by four SCI insecticides (indoxacarb, its bioactivated metabolite DCJW, metaflumizone, and RH3421). Mutations at Val787 affected SCI insecticide sensitivity in a manner that was independent of mutation-induced changes in slow inactivation gating. Sensitivity to inhibition by 10 μM indoxacarb was significantly increased in all three mutated channels, whereas sensitivity to inhibition by 10 μM metaflumizone was significantly reduced in Nav1.4/V787A channels and completely abolished in Nav1.4/V787K channels. The effects of Val787 mutations on metaflumizone were correlated with the hydrophobicity of the substituted amino acid rather than the extent of slow inactivation. None of the mutations at Val787 significantly affected the sensitivity to inhibition by DCJW or RH3421. These results demonstrate that the impact of mutations at Val787 on sodium channel inhibition by SCI insecticides depends on the specific insecticide examined and is independent of mutation-induced changes in slow inactivation gating. We propose that Val787 may be a unique determinant of metaflumizone binding. PMID:22983119

Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin.

PubMed

Nakamura, Toshio; Tonozuka, Takashi; Ide, Azusa; Yuzawa, Takayuki; Oguma, Keiji; Nishikawa, Atsushi

2008-02-22

Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal beta-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.

Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library.

PubMed

Houimel, Mehdi; Mazzucchelli, Luca

2013-01-01

Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for (125)I-hCCL11 binding to hCCR3 with IC(50) ranging from 3.5 to 9.7μM. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced peritoneal eosinophilia. The development of peptides inhibiting the interactions between hCCR3 and its chemokine ligands will facilitate the development of small peptides antagonists with the hope of ameliorating chronic inflammatory diseases in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

Menthol Binding and Inhibition of α7-Nicotinic Acetylcholine Receptors

PubMed Central

Ashoor, Abrar; Nordman, Jacob C.; Veltri, Daniel; Yang, Keun-Hang Susan; Al Kury, Lina; Shuba, Yaroslav; Mahgoub, Mohamed; Howarth, Frank C.; Sadek, Bassem; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

2013-01-01

Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca2+-dependent Cl− channels, since menthol inhibition remained unchanged by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing Ba2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner. PMID:23935840

Binding of /sup 125/I-hCG to rainbow trout (Salmo gairdneri) testis in vitro. [Human Chorionic Gonadotropin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schlaghecke, R.

1983-02-01

Homogenates of maturing rainbow trout testes show specific binding sites for /sup 125/I-labeled hCG (. /sup 125/I-labeled hCG). The binding is competitively inhibited by unlabeled hCG and by a hypophyseal extract of rainbow trout. It could be demonstrated that the tissue /sup 125/I-hCG binding specificity is restricted to the gonadal preparation. The trout testis was characterized by determining affinity and capacity from Scatchard plot analysis giving a high constant of dissociation Kd 3.65 x 10(-10)/M and a low binding capacity of 0.88 x 10(-15) M/mg tissue. The test system is markedly dependent on temperature, incubation-time, and pH. The maximum bindingmore » was found at 37 degrees during 2 hr of incubation in a buffer of pH 7.5.« less

Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein

PubMed Central

Rajsbaum, Ricardo; Albrecht, Randy A.; Wang, May K.; Maharaj, Natalya P.; Versteeg, Gijs A.; Nistal-Villán, Estanislao; García-Sastre, Adolfo; Gack, Michaela U.

2012-01-01

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production. PMID:23209422

Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein.

PubMed

Rajsbaum, Ricardo; Albrecht, Randy A; Wang, May K; Maharaj, Natalya P; Versteeg, Gijs A; Nistal-Villán, Estanislao; García-Sastre, Adolfo; Gack, Michaela U

2012-01-01

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.

Uptake of clostridium botulinum C3 exoenzyme into intact HT22 and J774A.1 cells.

PubMed

Rohrbeck, Astrid; von Elsner, Leonie; Hagemann, Sandra; Just, Ingo

2015-02-02

The Clostridium botulinum C3 exoenzyme selectively ADP-ribosylates low molecular weight GTP-binding proteins RhoA, B and C. This covalent modification inhibits Rho signaling activity, resulting in distinct actin cytoskeleton changes. Although C3 exoenzyme has no binding, the translocation domain assures that C3 enters cells and acts intracellularly. C3 uptake is thought to occur due to the high concentration of the C3 enzyme. However, recent work indicates that C3 is selectively endocytosed, suggesting a specific endocytotic pathway, which is not yet understood. In this study, we show that the C3 exoenzyme binds to cell surfaces and is internalized in a time-dependent manner. We show that the intermediate filament, vimentin, is involved in C3 uptake, as indicated by the inhibition of C3 internalization by acrylamide, a known vimentin disruption agent. Inhibition of C3 internalization was not observed by chemical inhibitors, like bafilomycin A, methyl-β-cyclodextrin, nocodazole or latrunculin B. Furthermore, the internalization of C3 exoenzyme was markedly inhibited in dynasore-treated HT22 cells. Our results indicate that C3 internalization depends on vimentin and does not depend strictly on both clathrin and caveolae.

Non-Watson–Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

PubMed Central

Tackett, Alan J.; Corey, David R.; Raney, Kevin D.

2002-01-01

Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications. PMID:11842106

Selective inhibition of CTCF binding by iAs directs TET-mediated reprogramming of 5-hydroxymethylation patterns in iAs-transformed cells

PubMed Central

Rea, Matthew; Gripshover, Tyler; Fondufe-Mittendorf, Yvonne

2017-01-01

Methylation at cytosine (5mC) is a fundamental epigenetic DNA modification recently associated with iAs-mediated carcinogenesis. In contrast, the role of 5-hydroxymethylcytosine (5hmC), the oxidation product of 5mC in iAs-mediated carcinogenesis is unknown. Here we assess the hydroxymethylome in iAs-transformed cells, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks. Moreover, this pathologic iAs-mediated carcinogenesis is characterized by a shift toward a higher hydroxymethylation pattern genome-wide. At specific promoters, hydroxymethylation correlated with increased gene expression. Furthermore, this increase in hydroxymethylation occurs concurrently with an upregulation of ten-eleven translocation (TET) enzymes that oxidize 5-methylcytosine (5mC) in DNA. To gain an understanding into how iAs might impact TET expression, we found that iAs inhibits the binding of CTCF at the proximal, weak CTCF binding sites of the TET1 and TET2 gene promoters and enhances CTCF binding at the stronger distal binding site. Further analyses suggest that this distal site acts as an enhancer, thus high CTCF occupancy at the enhancer region of TET1 and TET2 possibly drives their high expression in iAs-transformed cells. These results have major implications in understanding the impact of differential CTCF binding, genome architecture and its consequences in iAs-mediated pathogenesis. PMID:29175454

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

PubMed Central

Jensen, Jan K.; Malmendal, Anders; Schiøtt, Birgit; Skeldal, Sune; Pedersen, Katrine E.; Celik, Leyla; Nielsen, Niels Chr.; Andreasen, Peter A.; Wind, Troels

2006-01-01

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA–PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA–PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction. PMID:16813566

Genome-wide STAT3 binding analysis after histone deacetylase inhibition reveals novel target genes in dendritic cells

PubMed Central

Sun, Yaping; Iyer, Matthew; McEachin, Richard; Zhao, Meng; Wu, Yi-Mi; Cao, Xuhong; Oravecz-Wilson, Katherine; Zajac, Cynthia; Mathewson, Nathan; Wu, Shin-Rong Julia; Rossi, Corinne; Toubai, Tomomi; Qin, Zhaohui S.; Chinnaiya, Arul M.; Reddy, Pavan

2016-01-01

STAT3 is a master transcriptional regulator that plays an important role in the induction of both immune activation and immune tolerance in dendritic cells (DCs). The transcriptional targets of STAT3 in promoting DC activation are becoming increasingly understood; however, the mechanisms underpinning its role in causing DC suppression remain largely unknown. To determine the functional gene targets of STAT3, we compared the genome-wide binding of STAT3 using ChIP-seq coupled with gene expression microarrays to determine STAT3-dependent gene regulation in DCs after histone deacetylase (HDAC) inhibition. HDAC inhibition boosted the ability of STAT3 to bind to distinct DNA targets and regulate gene expression. Among the top 500 STAT3 binding sites, the frequency of canonical motifs was significantly higher than that of non-canonical motifs. Functional analysis revealed that after treatment with an HDAC inhibitor, the upregulated STAT3 target genes were those that were primarily the negative regulators of pro-inflammatory cytokines and those in the IL-10 signaling pathway. The downregulated STAT3-dependent targets were those involved in immune effector processes and antigen processing/presentation. The expression and functional relevance of these genes were validated. Specifically, functional studies confirmed that the upregulation of IL-10Ra by STAT3 contributed to the suppressive function of DCs following HDAC inhibition. PMID:27866206

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology.

PubMed

List, K; Høyer-Hansen, G; Rønne, E; Danø, K; Behrendt, N

1999-01-01

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.

Agonist and antagonist modulation of [35S]-GTPγS binding in transfected CHO cells expressing the neurotensin receptor

PubMed Central

Hermans, Emmanuel; Geurts, Muriel; Maloteaux, Jean-Marie

1997-01-01

The functional interaction of the cloned rat neurotensin receptor with intracellular G-proteins was investigated by studying the binding of the radiolabelled guanylyl nucleotide analogue [35S]-GTPγS induced by neurotensin to membranes prepared from transfected Chinese hamster ovary (CHO) cells. The agonist-induced binding of [35S]-GTPγS was only detected in the presence of NaCl in the incubation buffer. However, it was also demonstrated that the binding of [3H]-neurotensin to its receptor was inhibited by NaCl. In the presence of 50 mM NaCl, the binding of the labelled nucleotide was about 2 fold increased by stimulation with saturating concentrations of neurotensin (EC50 value of 2.3±0.9 nM). The stimulation of [35S]-GTPγS binding by neurotensin was mimicked by the stable analogue of neurotensin, JMV-449 (EC50 value of 1.7±0.4 nM) and the neurotensin related peptide neuromedin N (EC50 value of 21±6 nM). The NT-induced [35S]-GTPγS binding was competitively inhibited by SR48692 (pA2 value of 9.55±0.28), a non-peptide neurotensin receptor antagonist. SR48692 alone had no effect on the specific binding of [35S]-GTPγS. The response to neurotensin was found to be inhibited by the aminosteroid U-73122, a putative inhibitor of phospholipase C-dependent processes, indicating that this drug may act at the G-protein level. Taken together, these results constitute the first characterization of the exchange of guanylyl nucleotides at the G-protein level that is induced by the neuropeptide neurotensin after binding to its receptor. PMID:9283723

Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moldoveanu, Tudor; Gehring, Kalle; Green, Douglas R.

2009-01-15

Ca{sup 2+}-dependent cysteine proteases, calpains, regulate cell migration, cell death, insulin secretion, synaptic function and muscle homeostasis. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency. Despite the physiological importance of this interaction, the structural basis of calpain inhibition by calpastatin is unknown. Here we report the 3.0 A structure of Ca{sup 2+}-bound m-calpain in complex with the first calpastatin repeat, both from rat, revealing the mechanism of exclusive specificity. The structure highlights the complexity of calpain activation by Ca{sup 2+}, illustrating key residues in a peripheral domainmore » that serve to stabilize the protease core on Ca{sup 2+} binding. Fully activated calpain binds ten Ca{sup 2+} atoms, resulting in several conformational changes allowing recognition by calpastatin. Calpain inhibition is mediated by the intimate contact with three critical regions of calpastatin. Two regions target the penta-EF-hand domains of calpain and the third occupies the substrate-binding cleft, projecting a loop around the active site thiol to evade proteolysis.« less

Interplay of cis- and trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2.

PubMed

Absmeier, Eva; Becke, Christian; Wollenhaupt, Jan; Santos, Karine F; Wahl, Markus C

2017-01-02

RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing.

CD22 and Siglec-G regulate inhibition of B-cell signaling by sialic acid ligand binding and control B-cell tolerance.

PubMed

Nitschke, Lars

2014-09-01

CD22 and Siglec-G are two B-cell expressed members of the Siglec (sialic acid-binding immunoglobulin (Ig)-like lectin) family and are potent inhibitors of B-cell signaling. Genetic approaches have provided evidence that this inhibition of B-cell antigen receptor (BCR) signaling by Siglecs is dependent on ligand binding to sialic acids in specific linkages. The cis-ligand-binding activity of CD22 leads to homo-oligomer formation, which are to a large extent found in membrane domains that are distinct from those containing the BCR. In contrast, Siglec-G is recruited via sialic acid binding to the BCR. This interaction of Siglec-G with mIgM leads to an inhibitory function that seems to be specific for B-1 cells. Both CD22 and Siglec-G control B-cell tolerance and loss of these proteins, its ligands or its inhibitory pathways can increase the susceptibility for autoimmune diseases. CD22 is a target protein both in B-cell leukemias and lymphomas, as well as in B-cell mediated autoimmune diseases. Both antibodies and synthetic chemically modified sialic acids are currently tested to target Siglecs on B cells. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Optical sensors for therapeutic drug monitoring of antidepressants for a better medication adjustment

NASA Astrophysics Data System (ADS)

Krieg, Anne K.; Hess, Stefan; Gauglitz, Günter

2013-05-01

Therapeutic drug monitoring provides the attending physicians with detailed information on a patient's individual serum level especially during long-term medication. Due to the fact that each patient tolerates drugs or their metabolites differently a medication adjustment can reduce the number and intensity of noticeable side-effects. In particular, psychotropic drugs can cause unpleasant side-effects that affect a patient's life almost as much as the mental disease itself. The tricyclic antidepressants amitriptyline is commonly used for treatment of depressions and was selected for the development of an immunoassay using the direct optical sensor technique Reflectometric Interference Spectroscopy (RIfS). RIfS is a simple, robust and label-free method for direct monitoring of binding events on glass surfaces. Binding to the surface causes a shift of the interference spectrum by a change of the refractive index or physical thickness. This technique can be used for time-resolved observation of association and dissociation of amitriptyline (antigen) and a specific antibody using the binding inhibition test format. An amitriptyline derivative is immobilized on the sensor surface and a specific amount of antibodies can bind to the surface unless the binding is inhibited by free amitriptyline in a sample. No fluorescent label is needed making the whole assay less expensive than label-based methods. With this recently developed immunoassay amitriptyline concentrations in buffer (PBS) can easily be detected down to 500 ng/L.

Functional selection of a type IV pili-binding peptide that specifically inhibits Salmonella Typhi adhesion to/invasion of human monocytic cells.

PubMed

Wu, Hong-Yan; Zhang, Xiao-Lian; Pan, Qin; Wu, Jianguo

2005-11-01

Salmonella enterica serovar Typhi (S. Typhi) is an important pathogen which infects humans exclusively and causes typhoid or enteric fever. Recently it has been discovered that type IVB pili, encoded by the S. Typhi pil operon located in the major pathogenicity island, may be important in the pathogenesis of epidemic enteric fever. To further investigate the roles of type IVB pili of S. Typhi, a 12-mer peptide (RQERSSLSKPVV), binding to the structural protein PilS of the type IVB pili of S. Typhi, was isolated with a ribosome display system. This peptide was designated as peptide R. We found that peptide R inhibited adhesion to/invasion of human monocytic THP-1 cells by piliated S. Typhi bacteria, but had no effects on nonpiliated S. Typhi bacteria. A random 12-mer peptide, of size and solubility equal to peptide R, served as a control on the specificity of peptide R. The specific interaction and binding equilibrium between the 12-mer peptide R and PilS protein was determined by isothermal titration calorimetry (ITC) and a binding constant Ka determined to be between 0.4 x 10(5) and 2.2 x 10(5)L mol(-1). Our findings suggest that the type IV pili-binding peptide R holds potential as an antibacterial peptide effective against S. Typhi infections, both in terms of prevention and therapeutic treatment. The data further provide insights into the understanding of the pathogenic roles of the type IVB pili of S. Typhi.

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klein, M.; Canoll, P.D.; Musacchio, J.M.

1991-01-01

The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drugmore » has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.« less

Antisense RNA: effect of ribosome binding sites, target location, size, and concentration on the translation of specific mRNA molecules.

PubMed

Daugherty, B L; Hotta, K; Kumar, C; Ahn, Y H; Zhu, J D; Pestka, S

1989-01-01

A series of plasmids were constructed to generate RNA complementary to the beta-galactosidase messenger RNA under control of the phage lambda PL promoter. These plasmids generate anti-lacZ mRNA bearing or lacking a synthetic ribosome binding site adjacent to the lambda PL promoter and/or the lacZ ribosome binding site in reverse orientation. Fragments of lacZ DNA from the 5' and/or the 3' region were used in these constructions. When these anti-mRNA molecules were produced in Escherichia coli 294, maximal inhibition of beta-galactosidase synthesis occurred when a functional ribosome binding site was present near the 5' end of the anti-mRNA and the anti-mRNA synthesized was complementary to the 5' region of the mRNA corresponding to the lacZ ribosome binding site and/or the 5'-coding sequence. Anti-mRNAs producing maximal inhibition of beta-galactosidase synthesis exhibited an anti-lacZ mRNA:normal lacZ mRNA ratio of 100:1 or higher. Those showing lower levels of inhibition exhibited much lower anti-lacZ mRNA:normal lacZ mRNA ratios. A functional ribosome binding site at the 5'-end was found to decrease the decay rate of the anti-lacZ mRNAs. In addition, the incorporation of a transcription terminator just downstream of the antisense segment provided for more efficient inhibition of lacZ mRNA translation due to synthesis of smaller and more abundant anti-lacZ mRNAs. The optimal constructions produced undetectable levels of beta-galactosidase synthesis.

Selective Inhibition of the Human tie-1 Promoter with Triplex-Forming Oligonucleotides Targeted to Ets Binding Sites

PubMed Central

Hewett, Peter W; Daft, Emma L; Laughton, Charles A; Ahmad, Shakil; Ahmed, Asif; Murray, J Clifford

2006-01-01

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21–22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (Kd ~10−7 M) at 37 °C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction. PMID:16838069

Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to Ets binding sites.

PubMed

Hewett, Peter W; Daft, Emma L; Laughton, Charles A; Ahmad, Shakil; Ahmed, Asif; Murray, J Clifford

2006-01-01

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

Binding and degradation of 125I-gastrin by plasma membranes from homogenized rat gastric mucosa.

PubMed

Kleveland, P M; Waldum, H L

1986-06-01

Binding of 125I-gastrin to the 270-30,000 g fraction from homogenized rat oxyntic mucosa was studied. 'Specific' binding was calculated by subtracting the binding at excess cold gastrin from the binding with labelled gastrin (250 pM) only. At 30 degrees C specific binding rose rapidly to a short-lived maximum before falling gradually, whereas at 15 degrees C and 0 degree C specific binding rose gradually to a higher plateau level. The reduced binding at 30 degrees C could be caused by degradation of either the tracer or the binding site or by a combination of these two events. Degradation of 125I-gastrin was evaluated by trichloroacetic acid (TCA) precipitation, fast protein liquid chromatography, and binding to a gastrin antibody (immunoreactivity). The effect of incubation on the binding site was evaluated by preincubation of the homogenate fraction before adding gastrin. In separate studies, the proteolytic activity of the homogenate fraction was studied by TCA precipitation of radioactive casein. Different enzyme inhibitors tested were virtually ineffective in preventing gastrin and casein degradation. Only lowering the incubation temperature to 15 degrees C or lower could prevent this degradation. The reduced and transient binding of 125I-gastrin at 30 degrees C most probably reflects tracer degradation. Accordingly, the gastrin binding experiments were performed at 15 degrees C. Only homogenates from the oxyntic area of the stomach bound 125I-gastrin specifically and with a Kd of 0.8 nM (Scatchard analysis). However, micromolar concentrations of unlabelled gastrin were required to inhibit half maximal binding of the tracer. The tracer binding was unaffected by secretin, slightly reduced by a CCK-9 analogue, and more markedly reduced by pentagastrin.

n-Dodecyl β-D-maltoside specifically competes with general anesthetics for anesthetic binding sites.

PubMed

Xu, Longhe; Matsunaga, Felipe; Xi, Jin; Li, Min; Ma, Jingyuan; Liu, Renyu

2014-01-01

We recently demonstrated that the anionic detergent sodium dodecyl sulfate (SDS) specifically interacts with the anesthetic binding site in horse spleen apoferritin, a soluble protein which models anesthetic binding sites in receptors. This raises the possibility of other detergents similarly interacting with and occluding such sites from anesthetics, thereby preventing the proper identification of novel anesthetic binding sites. n-Dodecyl β-D-maltoside (DDM) is a non-ionic detergent commonly used during protein-anesthetic studies because of its mild and non-denaturing properties. In this study, we demonstrate that SDS and DDM occupy anesthetic binding sites in the model proteins human serum albumin (HSA) and horse spleen apoferritin and thereby inhibit the binding of the general anesthetics propofol and isoflurane. DDM specifically interacts with HSA (Kd = 40 μM) with a lower affinity than SDS (Kd = 2 μM). DDM exerts all these effects while not perturbing the native structures of either model protein. Computational calculations corroborated the experimental results by demonstrating that the binding sites for DDM and both anesthetics on the model proteins overlapped. Collectively, our results indicate that DDM and SDS specifically interact with anesthetic binding sites and may thus prevent the identification of novel anesthetic sites. Special precaution should be taken when undertaking and interpreting results from protein-anesthetic investigations utilizing detergents like SDS and DDM.

Pharmacological characterization of CCKB receptors in human brain: no evidence for receptor heterogeneity.

PubMed

Kinze, S; Schöneberg, T; Meyer, R; Martin, H; Kaufmann, R

1996-10-11

In this paper, cholecystokinin (CCK) B-type binding sites were characterized with receptor binding studies in different human brain regions (various parts of cerebral cortex, basal ganglia, hippocampus, thalamus, cerebellar cortex) collected from 22 human postmortem brains. With the exception of the thalamus, where no specific CCK binding sites were found, a pharmacological characterization demonstrated a single class of high affinity CCK sites in all brain areas investigated. Receptor densities ranged from 0.5 fmol/mg protein (hippocampus) to 8.4 fmol/mg protein (nucleus caudatus). These CCK binding sites displayed a typical CCKA binding profile as shown in competition studies by using different CCK-related compounds and non peptide CCK antagonists discriminating between CCKA and CCKB sites. The rank order of agonist or antagonist potency in inhibiting specific sulphated [propionyl-3H]cholecystokinin octapeptide binding was similar and highly correlated for the brain regions investigated as demonstrated by a computer-assisted analysis. Therefore it is concluded that CCKB binding sites in human cerebral cortex, basal ganglia, cerebellar cortex share identical ligand binding characteristics.

Integrin αvβ3 promotes infection by Japanese encephalitis virus.

PubMed

Fan, Wenchun; Qian, Ping; Wang, Dandan; Zhi, Xianwei; Wei, Yanming; Chen, Huanchun; Li, Xiangmin

2017-04-01

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is one of the major causes of viral encephalitis diseases worldwide. The JEV envelope protein facilitates viral entry, and its domain III contains an Arg-Gly-Asp (RGD) motif, that may modulate JEV entry through the RGD-binding integrin. In this study, the roles of integrin αv and β3 on the infection of JEV were evaluated. Reduced expression of integrin αv/β3 by special shRNA confers 2 to 4-fold inhibition of JEV replication in BHK-21 cells. Meanwhile, antibodies specific for integrin αv/β3 displayed ~58% and ~33% inhibition of JEV infectivity and RGD-specific peptides produced ~36% of inhibition. Expression of E protein and JEV RNA loads were clearly increased in CHO cells transfected with cDNA encoding human integrin β3. Moreover, integrin αv mediates JEV infection in viral binding stage of life cycle. Therefore, our study suggested that integrin αv and β3 serve as a host factor associated with JEV entry into the target cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

PARP-1 Controls the Adipogenic Transcriptional Program by PARylating C/EBPβ and Modulating Its Transcriptional Activity.

PubMed

Luo, Xin; Ryu, Keun Woo; Kim, Dae-Seok; Nandu, Tulip; Medina, Carlos J; Gupte, Rebecca; Gibson, Bryan A; Soccio, Raymond E; Yu, Yonghao; Gupta, Rana K; Kraus, W Lee

2017-01-19

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins mediated by PARP family members, such as PARP-1. Although PARylation has been studied extensively, few examples of definitive biological roles for site-specific PARylation have been reported. Here we show that C/EBPβ, a key pro-adipogenic transcription factor, is PARylated by PARP-1 on three amino acids in a conserved regulatory domain. PARylation at these sites inhibits C/EBPβ's DNA binding and transcriptional activities and attenuates adipogenesis in various genetic and cell-based models. Interestingly, PARP-1 catalytic activity drops precipitously during the first 48 hr of differentiation, corresponding to a release of C/EBPβ from PARylation-mediated inhibition. This promotes the binding of C/EBPβ at enhancers controlling the expression of adipogenic target genes and continued differentiation. Depletion or chemical inhibition of PARP-1, or mutation of the PARylation sites on C/EBPβ, enhances these early adipogenic events. Collectively, our results provide a clear example of how site-specific PARylation drives biological outcomes. Copyright © 2017 Elsevier Inc. All rights reserved.

Cargo selection by specific kinesin light chain 1 isoforms.

PubMed

Woźniak, Marcin J; Allan, Victoria J

2006-11-29

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures.

Cargo selection by specific kinesin light chain 1 isoforms

PubMed Central

Woźniak, Marcin J; Allan, Victoria J

2006-01-01

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures. PMID:17093494

Radioligand binding, autoradiographic and functional studies demonstrate tachykinin NK-2 receptors in dog urinary bladder.

PubMed

Mussap, C J; Stamatakos, C; Burcher, E

1996-10-01

Tachykinin receptors in the dog bladder were characterized using radioligand binding, functional and autoradiographic techniques. In detrusor muscle homogenates, specific binding of [125l]iodohistidyl neurokinin A (INKA) and [125l]Bolton Hunter eledoisin was reversible, saturable and, to a single class of sites of Kd, 3,6 and 27 nM, respectively. No specific binding of [125l]Bolton Hunter[Sar9, Met (O2)11] substance P occurred. INKA binding was reduced by the peptidase inhibitor bacitracin. The rank potency order of agonists competing for binding of both radioligands indicated interaction at NK-2 sites. NK-2-selective antagonists also competed for INKA binding, with SR 48968, GR 94800, MDL 29913 and the selective agonist [Lys5, MeLeu9, Nle10]-NKA(4-10) showing biphasic binding profiles. Autoradiographic studies revealed specific binding of INKA and [125l]Bolton Hunter eledoisin over detrusor muscle and small arteries. [125l]Bolton Hunter [Sar9, Met (O2)11] SP labeled the intima of arteries and arterioles, but not the detrusor muscle. Tachykinins contracted detrusor muscle strips, with potency order at the carbachol EC15 NKA = kassinin > [Lys5, MeLeu9, Nle10]-NKA(4-10) = neuropeptide gamma = neuropeptide K = NKB > > MDL 28564, with [Sar9, Met(O2)11]-SP ineffective. Shallow concentration-response curves, variable efficacies and inhibition by atropine and mepyramine suggest that other mechanisms may influence contractile responses. Responses to [Lys5, MeLeu9, Nle10]-NKA(4-10) were inhibited competitively by MDL 29913 and MEN 10207 (pA2 values: 6.4 and 5.3, respectively). Antagonism by SR 48968 and GR 94800 was noncompetitive (both pK8 values 8.9). In summary, NK-2-preferring ligands showed superior potency as both binding competitors and contractile agonists, demonstrating that NK-2 receptors mediate detrusor muscle contraction, similar to the human detrusor. Tachykinins may play important roles in the micturition reflex and in regulating detrusor muscle blood flow in the dog.

Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication.

PubMed

Mukherjee, Sourav; Weiner, Warren S; Schroeder, Chad E; Simpson, Denise S; Hanson, Alicia M; Sweeney, Noreena L; Marvin, Rachel K; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J; Frick, David N

2014-10-17

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.

Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha.

PubMed

De Marco, L; Mazzucato, M; Masotti, A; Ruggeri, Z M

1994-03-04

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

Inhibition of P-fimbriated Escherichia coli adhesion by multivalent galabiose derivatives studied by a live-bacteria application of surface plasmon resonance.

PubMed

Salminen, Annika; Loimaranta, Vuokko; Joosten, John A F; Khan, A Salam; Hacker, Jörg; Pieters, Roland J; Finne, Jukka

2007-09-01

Uropathogenic P-fimbriated Escherichia coli adheres to host cells by specific adhesins recognizing galabiose (Galalpha1-4Gal)-containing structures on cell surfaces. In search of agents inhibiting this first step of infection, the inhibition potency of a set of synthetic mono- and multivalent galabiose compounds was evaluated. In order to mimic the flow conditions of natural infections, a live-bacteria application of surface plasmon resonance (SPR) was established. For the measurement of the binding of E. coli to a surface containing galabiose, live bacteria were injected over the flow cell, and the inhibition of adhesion caused by the galabiose inhibitors was recorded. Quantitative binding data were recorded in real-time for each inhibitor. The results were compared with those of conventional static haemagglutination and ELISA-based cell adhesion assays. Compared with the Gram-positive Streptococcus suis bacteria, which also bind to galabiose and whose binding inhibition is strongly dependent on the multivalency of the inhibitor, E. coli inhibition was only moderately affected by the valency. However, a novel octavalent compound was found to be the most effective inhibitor of E. coli PapG(J96) adhesion, with an IC50 value of 2 microM. Measurement of bacterial adhesion by SPR is an efficient way to characterize the adhesion of whole bacterial cells and allows the characterization of the inhibitory potency of adhesion inhibitors under dynamic flow conditions. Under these conditions, multivalency increases the anti-adhesion potency of galabiose-based inhibitors of P-fimbriated E. coli adhesion and provides a promising approach for the design of high-affinity anti-adhesion agents.

Demonstration of isoleucine 199 as a structural determinant for the selective inhibition of human monoamine oxidase B by specific reversible inhibitors.

PubMed

Hubálek, Frantisek; Binda, Claudia; Khalil, Ashraf; Li, Min; Mattevi, Andrea; Castagnoli, Neal; Edmondson, Dale E

2005-04-22

Several reversible inhibitors selective for human monoamine oxidase B (MAO B) that do not inhibit MAO A have been described in the literature. The following compounds: 8-(3-chlorostyryl)caffeine, 1,4-diphenyl-2-butene, and trans,trans-farnesol are shown to inhibit competitively human, horse, rat, and mouse MAO B with K(i) values in the low micromolar range but are without effect on either bovine or sheep MAO B or human MAO A. In contrast, the reversible competitive inhibitor isatin binds to all known MAO B and MAO A with similar affinities. Sequence alignments and the crystal structures of human MAO B in complex with 1,4-diphenyl-2-butene or with trans,trans-farnesol provide molecular insights into these specificities. These inhibitors span the substrate and entrance cavities with the side chain of Ile-199 rotated out of its normal conformation suggesting that Ile-199 is gating the substrate cavity. Ile-199 is conserved in all known MAO B sequences except bovine MAO B, which has Phe in this position (the sequence of sheep MAO B is unknown). Phe is conserved in the analogous position in MAO A sequences. The human MAO B I199F mutant protein of MAO B binds to isatin (K(i) = 3 microM) but not to the three inhibitors listed above. The crystal structure of this mutant demonstrates that the side chain of Phe-199 interferes with the binding of those compounds. This suggests that the Ile-199 "gate" is a determinant for the specificity of these MAO B inhibitors and provides a molecular basis for the development of MAO B-specific reversible inhibitors without interference with MAO A function in neurotransmitter metabolism.

The structural basis for RNA specificity and Ca2+ inhibition of an RNA-dependent RNA polymerase.

PubMed

Salgado, Paula S; Makeyev, Eugene V; Butcher, Sarah J; Bamford, Dennis H; Stuart, David I; Grimes, Jonathan M

2004-02-01

The RNA-dependent RNA polymerase of bacteriophage phi6 transcribes mRNA from the three segments of the dsRNA viral genome. We have cocrystallized RNA oligonucleotides with the polymerase, revealing the mode of binding of RNA templates. This binding is somewhat different from that previously seen for DNA oligomers, leading to additional RNA-protein hydrogen bonds, consistent with a preference for RNA. Activation of the RNA/polymerase complex by the addition of substrate and Mg2+ initiates a single round of reaction within the crystal to form a dead-end complex that partially collapses within the enzyme active site. By replacing Mg2+ with Ca2+, we have been able to capture the inhibited complex which shows distortion that explains the structural basis for the inhibition of such polymerases by Ca2+.

The binding of Varp to VAMP7 traps VAMP7 in a closed, fusogenically inactive conformation

PubMed Central

Schäfer, Ingmar B.; Hesketh, Geoffrey G.; Bright, Nicholas A.; Gray, Sally R.; Pryor, Paul R.; Evans, Philip R; Luzio, J. Paul; Owen, David J.

2012-01-01

SNAREs provide energy and specificity to membrane fusion events. Fusogenic trans-SNARE complexes are assembled from Q-SNAREs embedded in one membrane and an R–SNARE embedded in the other. Regulation of membrane fusion events is crucial for intracellular trafficking. We identify the endosomal protein Varp as an R-SNARE-binding regulator of SNARE complex formation. Varp co-localises with and binds to VAMP7, an R-SNARE involved in both endocytic and secretory pathways. We present the structure of the second ankyrin repeat domain of mammalian Varp in complex with the cytosolic portion of VAMP7. The VAMP7 SNARE motif is trapped between Varp and the VAMP7 longin domain and hence Varp kinetically inhibits VAMP7’s ability to form SNARE complexes. This inhibition will be increased when Varp can also bind to other proteins present on the same membrane as the VAMP7 such as Rab32:GTP. PMID:23104059

Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane

PubMed Central

Novakovic, Predrag; Huang, Yanyun Y.; Lockerbie, Betty; Shahriar, Farshid; Kelly, John; Gordon, John R.; Middleton, Dorothy M.; Loewen, Matthew E.; Kidney, Beverly A.; Simko, Elemir

2015-01-01

F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization. PMID:25852227

Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

USGS Publications Warehouse

Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

2004-01-01

Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2

PubMed Central

White, Rebekah R.; Shan, Siqing; Rusconi, Christopher P.; Shetty, Geetha; Dewhirst, Mark W.; Kontos, Christopher D.; Sullenger, Bruce A.

2003-01-01

Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factor-mediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent. PMID:12692304

Diagnosis of Plasmodium falciparum infection in man: detection of parasite antigens by ELISA*

PubMed Central

Mackey, L. J.; McGregor, I. A.; Paounova, N.; Lambert, P. H.

1982-01-01

An ELISA method has been developed for the diagnosis of Plasmodium falciparum infection in man. Parasites from in vitro cultures of P. falciparum were used as source of antigen for the solid phase and the source of specific antibody was immune Gambian sera; binding of antibody in antigen-coated wells was registered by means of alkaline phosphatase-conjugated anti-human IgG. Parasites were detected on the basis of inhibition of antibody-binding. The test was applied to the detection of parasites in human red blood cells (RBC) from in vitro cultures of P. falciparum and in RBC from infected Gambians; RBC from 100 Geneva blood donors served as normal, uninfected controls. In titration experiments, the degree of antibody-binding inhibition correlated with the number of parasites in the test RBC. Parasites were detected at a level of 8 parasites/106 RBC. Samples of RBC were tested from 126 Gambians with microscopically proven infection; significant antibody-binding inhibition was found in 86% of these cases, where parasitaemia ranged from 10 to 125 000/μl of blood. The presence of high-titre antibody in the test preparations was found to reduce the sensitivity of parasite detection in infected RBC from in vitro cultures mixed with equal volumes of different antibody-containing sera. The sensitivity was restored in most cases by recovering the RBC by centrifugation before testing. In a preliminary experiment, there was no significant difference in antibody-binding inhibition using fresh infected RBC and RBC dried on filter-paper and recovered by elution, although there was greater variation in the latter samples. PMID:7044589

Identification of two H3-histamine receptor subtypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

West, R.E. Jr.; Zweig, A.; Shih, N.Y.

The H3-histamine receptor provides feedback inhibition of histamine synthesis and release as well as inhibition of other neurotransmitter release. We have characterized this receptor by radioligand binding studies with the H3 agonist N alpha-(3H)methylhistamine ((3H)NAMHA). The results of (3H)NAMHA saturation binding and NAMHA inhibition of (3H)NAMHA binding were consistent with an apparently single class of receptors (KD = 0.37 nM, Bmax = 73 fmol/mg of protein) and competition assays with other agonists and the antagonists impromidine and dimaprit disclosed only a single class of sites. In contrast, inhibition of (3H)NAMHA binding by the specific high affinity H3 antagonist thioperamide revealedmore » two classes of sites (KiA = 5 nM, BmaxA = 30 fmol/mg of protein; KiB = 68 nM, BmaxB = 48 fmol/mg of protein). Burimamide, another antagonist that, like thioperamide, contains a thiourea group, likewise discriminated between two classes of sites. In addition to differences between some antagonist potencies for the two receptors, there is a differential guanine nucleotide sensitivity of the two. The affinity of the H3A receptor for (3H) NAMHA was reduced less than 2-fold, whereas (3H)NAMHA binding to the H3B receptor was undetectable in the presence of guanosine 5'-O-(3-thiotriphosphate). The distinction between H3A and H3B receptor subtypes, the former a high affinity and the latter a low affinity thioperamide site, draws support from published in vitro data.« less

Tumor necrosis factor interaction with gold nanoparticles

NASA Astrophysics Data System (ADS)

Tsai, De-Hao; Elzey, Sherrie; Delrio, Frank W.; Keene, Athena M.; Tyner, Katherine M.; Clogston, Jeffrey D.; Maccuspie, Robert I.; Guha, Suvajyoti; Zachariah, Michael R.; Hackley, Vincent A.

2012-05-01

We report on a systematic investigation of molecular conjugation of tumor necrosis factor-α (TNF) protein onto gold nanoparticles (AuNPs) and the subsequent binding behavior to its antibody (anti-TNF). We employ a combination of physical and spectroscopic characterization methods, including electrospray-differential mobility analysis, dynamic light scattering, polyacrylamide gel electrophoresis, attenuated total reflectance-Fourier transform infrared spectroscopy, fluorescence assay, and enzyme-linked immunosorbent assay. The native TNF used in this study exists in the active homotrimer configuration prior to conjugation. After binding to AuNPs, the maximum surface density of TNF is (0.09 +/- 0.02) nm-2 with a binding constant of 3 × 106 (mol L-1)-1. Dodecyl sulfate ions induce desorption of monomeric TNF from the AuNP surface, indicating a relatively weak intermolecular binding within the AuNP-bound TNF trimers. Anti-TNF binds to both TNF-conjugated and citrate-stabilized AuNPs, showing that non-specific binding is significant. Based on the number of anti-TNF molecules adsorbed, a substantially higher binding affinity was observed for the TNF-conjugated surface. The inclusion of thiolated polyethylene glycol (SH-PEG) on the AuNPs inhibits the binding of anti-TNF, and the amount of inhibition is related to the number ratio of surface bound SH-PEG to TNF and the way in which the ligands are introduced. This study highlights the challenges in quantitatively characterizing complex hybrid nanoscale conjugates, and provides insight on TNF-AuNP formation and activity.We report on a systematic investigation of molecular conjugation of tumor necrosis factor-α (TNF) protein onto gold nanoparticles (AuNPs) and the subsequent binding behavior to its antibody (anti-TNF). We employ a combination of physical and spectroscopic characterization methods, including electrospray-differential mobility analysis, dynamic light scattering, polyacrylamide gel electrophoresis, attenuated total reflectance-Fourier transform infrared spectroscopy, fluorescence assay, and enzyme-linked immunosorbent assay. The native TNF used in this study exists in the active homotrimer configuration prior to conjugation. After binding to AuNPs, the maximum surface density of TNF is (0.09 +/- 0.02) nm-2 with a binding constant of 3 × 106 (mol L-1)-1. Dodecyl sulfate ions induce desorption of monomeric TNF from the AuNP surface, indicating a relatively weak intermolecular binding within the AuNP-bound TNF trimers. Anti-TNF binds to both TNF-conjugated and citrate-stabilized AuNPs, showing that non-specific binding is significant. Based on the number of anti-TNF molecules adsorbed, a substantially higher binding affinity was observed for the TNF-conjugated surface. The inclusion of thiolated polyethylene glycol (SH-PEG) on the AuNPs inhibits the binding of anti-TNF, and the amount of inhibition is related to the number ratio of surface bound SH-PEG to TNF and the way in which the ligands are introduced. This study highlights the challenges in quantitatively characterizing complex hybrid nanoscale conjugates, and provides insight on TNF-AuNP formation and activity. Electronic supplementary information (ESI) available: Experimental procedures, instrumentation, materials and calculations. See DOI: 10.1039/c2nr30415e

Binding of perlecan to transthyretin in vitro.

PubMed Central

Smeland, S; Kolset, S O; Lyon, M; Norum, K R; Blomhoff, R

1997-01-01

Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein-glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions. PMID:9307034

MuSK IgG4 autoantibodies cause myasthenia gravis by inhibiting binding between MuSK and Lrp4

PubMed Central

Huijbers, Maartje G.; Zhang, Wei; Klooster, Rinse; Niks, Erik H.; Friese, Matthew B.; Straasheijm, Kirsten R.; Thijssen, Peter E.; Vrolijk, Hans; Plomp, Jaap J.; Vogels, Pauline; Losen, Mario; Van der Maarel, Silvère M.; Burden, Steven J.; Verschuuren, Jan J.

2013-01-01

Myasthenia gravis (MG) is a severely debilitating autoimmune disease that is due to a decrease in the efficiency of synaptic transmission at neuromuscular synapses. MG is caused by antibodies against postsynaptic proteins, including (i) acetylcholine receptors, the neurotransmitter receptor, (ii) muscle-specific kinase (MuSK), a receptor tyrosine kinase essential for the formation and maintenance of neuromuscular synapses, and (iii) low-density lipoprotein receptor-related protein 4 (Lrp4), which responds to neural Agrin by binding and stimulating MuSK. Passive transfer studies in mice have shown that IgG4 antibodies from MuSK MG patients cause disease without requiring complement or other immune components, suggesting that these MuSK antibodies cause disease by directly interfering with MuSK function. Here we show that pathogenic IgG4 antibodies to MuSK bind to a structural epitope in the first Ig-like domain of MuSK, prevent binding between MuSK and Lrp4, and inhibit Agrin-stimulated MuSK phosphorylation. In contrast, these IgG4 antibodies have no direct effect on MuSK dimerization or MuSK internalization. These results provide insight into the unique pathogenesis of MuSK MG and provide clues toward development of specific treatment options. PMID:24297891

Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization

NASA Technical Reports Server (NTRS)

Barmina, O. Y.; Walling, H. W.; Fiacco, G. J.; Freije, J. M.; Lopez-Otin, C.; Jeffrey, J. J.; Partridge, N. C.

1999-01-01

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus*

PubMed Central

Hendricks, Gabriel L.; Weirich, Kim L.; Viswanathan, Karthik; Li, Jing; Shriver, Zachary H.; Ashour, Joseph; Ploegh, Hidde L.; Kurt-Jones, Evelyn A.; Fygenson, Deborah K.; Finberg, Robert W.; Comolli, James C.; Wang, Jennifer P.

2013-01-01

Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains. PMID:23362274

Interaction of Diuron and Related Substituted Phenylureas with the Ah Receptor Pathway

PubMed Central

Zhao, Bin; Baston, David S.; Hammock, Bruce; Denison, Michael S.

2011-01-01

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of structurally diverse chemicals, including the ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin. Here, we have examined the ability of diuron, a widely used herbicide, and several structurally related substituted phenylureas to bind to and activate/inhibit the AhR and AhR signal transduction. Diuron induced CYP1A1 mRNA levels in mouse hepatoma (Hepa1c1c7) cells and AhR-dependent luciferase reporter gene expression in stably transfected mouse, rat, guinea pig, and human cell lines. In addition, ligand binding and gel retardation analysis demonstrated the ability of diuron to competitively bind to and stimulate AhR transformation and DNA binding in vitro and in intact cells. Several structurally related substituted phenylureas competitively bound to the guinea pig hepatic cytosolic AhR, inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced AhR-dependent luciferase reporter gene expression in a species-specific manner and stimulated AhR transformation and DNA binding, consistent with their role as partial AhR agonists. These results demonstrate not only that diuron and related substituted phenylureas are AhR ligands but also that exposure to these chemicals could induce/inhibit AhR-dependent biological effects. PMID:16788953

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lever, J.R.; Scheffel, U.; Stathis, M.

Analogues of diprenorphine (DPN) having C6-O-iodoallyl (O-IA-DPN) and N-iodoallyl (N-IA-DPN) substituents can be I-125 labeled in good yield with high specific activity by radioiododestannylation. When tested in vitro against [H-3]-DPN in rat brain membranes, the apparent affinity (Ki) of O-IA-DPN (1.35 nM) proved 17-fold stronger than that of N-IA-DPN (23.4 nM). Against selective [H-3]-ligands, O-IA-DPN showed high apparent affinities for {mu}(1.9 nM), {gamma}(1.1 nM) and {kappa}(0.9 nM) sites. Consistent with the low apparent affinity in vitro, [I-125]-N-IA- DPN did not allow localization of cerebral opioid receptors after i.v. administration to mice. By contrast, [I-125]-O-IA-DPN exhibited a regional brain distribution whichmore » reflects binding to multiple opioid receptors. The highest radioactivity concentrations were in superior colliculi, hypothalamus, olfactory tubercles, thalamus and striatum. Peak levels (2.5-3.5 %ID/g) were maintained over the first 60 min. At all times, the lowest levels of radioactivity were in the cerebellum. Binding in vivo was saturable by O-IA-DPN, was blocked by (-)- but not by (+)-naloxone, and was inhibited by naltrexone in dose-dependent fashion. Specific binding was 83-93% for all tissues except cerebellum, where 50% blockade was noted with naltrexone (5.0 mg/kg). Using naltrexone blockade to define non-specific binding, the highest ratio of specific to non-specific binding (> 14 to 1) was noted for superior colliculi at 60 min. Inhibition studies with drugs selective for {mu}, {gamma} or {kappa} sites established that multiple opioid receptors are labeled. [123I]-O-IA-DPN has been prepared (84%, >2400 mCi/{mu}mol), and allows visualization of opioid receptors in mouse brain by ex vivo autoradiography. Together, these results suggest that [123I]-O-IA-DPN is suitable for SPECT studies of multiple opioid receptors.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brodde, O.E.; Leifert, F.J.; Krehl, H.J.

We determined the amount of beta 1- and beta 2-adrenoceptors in right and left atria and ventricles of rabbits. For this purpose inhibition of specific (-)-/sup 3/H-dihydroalprenolol ((-)-/sup 3/H-DHA) binding (5 nM) by beta 1-selective (practolol, metoprolol) and beta 2-selective (zinterol, IPS 339) adrenergic drugs was determined and analyzed by pseudo-Scatchard (Hofstee) plots. For both atria, inhibition of binding by the four selective beta-adrenergic drugs resulted in non-linear Hofstee plots, suggesting the coexistence of both beta-adrenoceptor subtypes. From these plots we calculated a beta 1:beta 2-adrenoceptor ratio of 72:28 for the right atrium and of 82:18 for the left. Inmore » contrast, only a very small amount of beta 2-adrenoceptors (approximately 5-7% of the total beta-adrenoceptor population) could be detected in the ventricles. For comparison we analyzed the inhibition of specific (-)-/sup 3/H-DHA binding in tissues with homogeneous population of beta-adrenoceptors (beta 1:guinea pig left ventricle; beta 2: cerebellum of mature rats). For both tissues the four selective beta-adrenergic drugs showed linear Hofstee plots, demonstrating that in tissues with homogeneous beta-receptor population interaction of each drug with the receptor followed simple mass-action kinetics. We conclude that beta 1- and beta 2-adrenoceptors coexist in rabbit atria while the ventricles are predominantly endowed the beta 1-adrenoceptors.« less

Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation

PubMed Central

Meeks, Shannon L.; Healey, John F.; Parker, Ernest T.; Barrow, Rachel T.

2007-01-01

The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/Phe2200 phospholipid binding β-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 β-sandwich. MAbs from groups A, AB, and B inhibit the binding of fVIIIa to phospholipid membranes. Group BC was the most common group and displayed the highest specific fVIII inhibitor activities. MAbs in this group are type II inhibitors that inhibit the activation of fVIII by either thrombin or factor Xa and poorly inhibit the binding of fVIII to phospholipid membranes or von Willebrand factor (VWF). Group BC MAbs are epitopically and mechanistically distinct from the extensively studied group C MAb, ESH8. These results reveal the structural and functional complexity of the anti-C2 domain antibody response and indicate that interference with fVIII activation is a major attribute of the inhibitor landscape. PMID:17848617

Synergistic Regulation of Competence Development in Bacillus subtilis by Two Rap-Phr Systems† ‡

PubMed Central

Bongiorni, Cristina; Ishikawa, Shu; Stephenson, Sophie; Ogasawara, Naotake; Perego, Marta

2005-01-01

The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is involved in the regulation of competence to DNA transformation. The ComA response regulator and transcription factor for initiation of competence development is the target of RapF. Specific binding of RapF to the carboxy-terminal DNA-binding domain of ComA inhibits the response regulator's ability to bind its target DNA promoters. The PhrF C-terminal pentapeptide, QRGMI, inhibits RapF activity. The activity of RapF and PhrF in regulating competence development is analogous to the previously described activity of RapC and PhrC (L. J. Core and M. Perego, Mol. Microbiol. 49:1509-1522, 2003). In fact, the RapF and PhrF pair of proteins acts synergistically with RapC and PhrC in the overall regulation of the ComA transcription factor. Since the transcription of the RapC- and RapF-encoding genes is positively regulated by their own target ComA, an autoregulatory circuit must exist for the competence transcription factor in order to modulate its activity. PMID:15968044

Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boone, Christopher D.; Tu, Chingkuang; McKenna, Robert, E-mail: rmckenna@ufl.edu

The structure of human carbonic anhydrase II in complex with cholate has been determined to 1.54 Å resolution. Elucidation of the novel inhibition mechanism of cholate will aid in the development of a nonsulfur-containing, isoform-specific therapeutic agent. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO{sub 2} into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes tomore » mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Å resolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy and osteoporosis.« less

Anti-idiotypic antibodies to poliovirus antibodies in commercial immunoglobulin preparations, human serum, and milk.

PubMed

Hahn-Zoric, M; Carlsson, B; Jeansson, S; Ekre, H P; Osterhaus, A D; Roberton, D; Hanson, L A

1993-05-01

Our previous studies have suggested that fetal antibody production can be induced by maternal antiidiotypic antibodies transferred to the fetus via the placenta. We tested commercial Ig, sera, and milk for the presence of anti-idiotypic antibodies to poliovirus type 1, using affinity chromatography combined with ELISA systems and virus neutralization techniques. Our results indicate that commercial Ig, serum, and milk samples contain antibodies recognizing idiotypic determinants on antibodies to poliovirus. Several lines of evidence support this conclusion. Thus, in an ELISA with poliovirus as a solid phase, binding of specific antibodies could be inhibited by addition of an eluate from the Ig preparation containing anti-idiotypic antibodies against poliovirus type 1. Also, antiidiotypic antibodies from pooled human Ig, serum, and colostrum samples against poliovirus bound directly to solid-phase-attached MAb against poliovirus type 1. In addition, in a competitive inhibition ELISA, where antiidiotypic antibodies isolated from the Ig preparation competed with the poliovirus antigen for binding to monoclonal or polyclonal idiotypic antibodies on the solid phase, inhibition of antigen binding was seen at low antigen concentrations. When single-donor serum or milk was used, this inhibition was even more pronounced and could be demonstrated at almost all antigen concentrations. The finding that anti-idiotypes are present in maternal serum and milk imply, in agreement with our previous studies, that anti-idiotypes may actively induce a specific immune response in the fetus without previous exposure to the antigen by being transferred across the placenta or by being passively transferred to the newborn via mother's milk.

Chicken galectin-1B inhibits Newcastle disease virus adsorption and replication through binding to hemagglutinin-neuraminidase (HN) glycoprotein.

PubMed

Sun, Junfeng; Han, Zongxi; Qi, Tianming; Zhao, Ran; Liu, Shengwang

2017-12-08

Galectin-1 is an important immunoregulatory factor and can mediate the host-pathogen interaction via binding glycans on the surface of various viruses. We previously reported that avian respiratory viruses, including lentogenic Newcastle disease virus (NDV), can induce up-regulation of chicken galectin (CG)-1B in the primary target organ. In this study, we investigated whether CG-1B participated in the infectious process of NDV in chickens. We demonstrated that velogenic NDV induced up-regulation of CG-1B in target organs. We also found that CG-1B directly bound to NDV virions and inhibited their hemagglutination activity in vitro We confirmed that CG-1B interacted with NDV hemagglutinin-neuraminidase (HN) glycoprotein, in which the specific G4 N -glycans significantly contributed to the interaction between CG-1B and HN glycoprotein. The presence of extracellular CG-1B, rather than the internalization process, inhibited adsorption of NDV. The interaction between intracellular CG-1B and NDV HN glycoproteins inhibited cell-surface expression of HN glycoprotein and reduced the titer of progeny virus in NDV-infected DF-1 cells. Significantly, the replication of parental and HN glycosylation mutant viruses in CG-1B knockdown and overexpression cells demonstrated that the replication of NDV was correlated with the expression of CG-1B in a specific glycan-dependent manner. Collectively, our results indicate that CG-1B has anti-NDV activity by binding to N -glycans on HN glycoprotein. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowler, C.J.; Ahlgren, P.C.; O'Neill, C.

IMR-32 and SK-N-MC cells were found to contain ({sup 3}H)quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable ({sup 3}H)8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC{sub 50} value of about 50 {mu}M in both cases. Pirenzepine inhibited the carbachol stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC{sub 50} values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT{sub 1A} receptor agonistmore » 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA{sub 2} values of 5.78 (IMR-32) and 5.61 (SK-N-MC). The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.« less

Trim25 Is an RNA-Specific Activator of Lin28a/TuT4-Mediated Uridylation.

PubMed

Choudhury, Nila Roy; Nowak, Jakub S; Zuo, Juan; Rappsilber, Juri; Spoel, Steven H; Michlewski, Gracjan

2014-11-20

RNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis, thereby providing an additional level of specificity.

LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

PubMed

Clayton, R N; Shakespear, R A; Marshall, J C

1978-06-01

Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

Warfarin traps human vitamin K epoxide reductase in an intermediate state during electron transfer

PubMed Central

Shen, Guomin; Cui, Weidong; Zhang, Hao; Zhou, Fengbo; Huang, Wei; Liu, Qian; Yang, Yihu; Li, Shuang; Bowman, Gregory R.; Sadler, J. Evan; Gross, Michael L.; Li, Weikai

2017-01-01

Although warfarin is the most widely used anticoagulant worldwide, the mechanism by which warfarin inhibits its target, human vitamin K epoxide reductase (hVKOR), remains unclear. Here we show that warfarin blocks a dynamic electron-transfer process in hVKOR. A major fraction of cellular hVKOR is at an intermediate redox state of this process containing a Cys51-Cys132 disulfide, a characteristic accommodated by a four-transmembrane-helix structure of hVKOR. Warfarin selectively inhibits this major cellular form of hVKOR, whereas disruption of the Cys51-Cys132 disulfide impairs warfarin binding and causes warfarin resistance. Relying on binding interactions identified by cysteine alkylation footprinting and mass spectrometry coupled with mutagenesis analysis, we are able to conduct structure simulations to reveal a closed warfarin-binding pocket stabilized by the Cys51-Cys132 linkage. Understanding the selective warfarin inhibition of a specific redox state of hVKOR should enable the rational design of drugs that exploit the redox chemistry and associated conformational changes in hVKOR. PMID:27918545

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.

PubMed

Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui; Falconieri, Veronica; Rao, Prashant; Merk, Alan; Eng, Edward T; Raczkowski, Ashleigh M; Fox, Tara; Earl, Lesley A; Patel, Dinshaw J; Subramaniam, Sriram

2017-10-05

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition. Published by Elsevier Inc.

Amyloid-β nanotubes are associated with prion protein-dependent synaptotoxicity

PubMed Central

Nicoll, Andrew J.; Panico, Silvia; Freir, Darragh B.; Wright, Daniel; Terry, Cassandra; Risse, Emmanuel; Herron, Caroline E.; O’Malley, Tiernan; Wadsworth, Jonathan D. F.; Farrow, Mark A.; Walsh, Dominic M.; Saibil, Helen R.; Collinge, John

2013-01-01

Growing evidence suggests water-soluble, non-fibrillar forms of amyloid-β protein (Aβ) have important roles in Alzheimer’s disease with toxicities mimicked by synthetic Aβ1–42. However, no defined toxic structures acting via specific receptors have been identified and roles of proposed receptors, such as prion protein (PrP), remain controversial. Here we quantify binding to PrP of Aβ1–42 after different durations of aggregation. We show PrP-binding and PrP-dependent inhibition of long-term potentiation (LTP) correlate with the presence of protofibrils. Globular oligomers bind less avidly to PrP and do not inhibit LTP, whereas fibrils inhibit LTP in a PrP-independent manner. That only certain transient Aβ assemblies cause PrP-dependent toxicity explains conflicting reports regarding the involvement of PrP in Aβ-induced impairments. We show that these protofibrils contain a defined nanotubular structure with a previously unidentified triple helical conformation. Blocking the formation of Aβ nanotubes or their interaction with PrP might have a role in treatment of Alzheimer’s disease. PMID:24022506

Interaction of SR 33557 with skeletal muscle calcium channel blocker receptors in the baboon: characterization of its binding sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sol-Rolland, J.; Joseph, M.; Rinaldi-Carmona, M.

1991-05-01

A procedure for the isolation of primate skeletal microsomal membranes was initiated. Membranes exhibited specific enzymatic markers such as 5'-nucleotidase, Ca{sup 2}{sup +},Mg({sup 2}{sup +})-adenosine triphosphatase and an ATP-dependent calcium uptake. Baboon skeletal microsomes bound specifically with high-affinity potent Ca{sup 2}{sup +} channel blockers such as dihydropyridine, phenylalkylamine and benzothiazepine derivatives. Scatchard analysis of equilibrium binding assays with ({sup 3}H)(+)-PN 200-110, ({sup 3}H)(-)-desmethoxyverapamil (( {sup 3}H)(-)-D888) and ({sup 3}H)-d-cis-dilitiazem were consistent with a single class of binding sites for the three radioligands. The pharmacological profile of SR 33557, an original compound with calcium antagonist properties, was investigated using radioligand bindingmore » studies. SR 33557 totally inhibited the specific binding of the three main classes of Ca{sup 2}{sup +} channel effectors and interacted allosterically with them. In addition, SR 33557 bound with high affinity to a homogeneous population of binding sites in baboon skeletal muscle.« less

Solubilization and purification of melatonin receptors from lizard brain.

PubMed

Rivkees, S A; Conron, R W; Reppert, S M

1990-09-01

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

Volatile anesthetics compete for common binding sites on bovine serum albumin: a 19F-NMR study.

PubMed Central

Dubois, B W; Cherian, S F; Evers, A S

1993-01-01

There is controversy as to the molecular nature of volatile anesthetic target sites. One proposal is that volatile anesthetics bind directly to hydrophobic binding sites on certain sensitive target proteins. Consistent with this hypothesis, we have previously shown that a fluorinated volatile anesthetic, isoflurane, binds saturably [Kd (dissociation constant) = 1.4 +/- 0.2 mM, Bmax = 4.2 +/- 0.3 sites] to fatty acid-displaceable domains on serum albumin. In the current study, we used 19F-NMR T2 relaxation to examine whether other volatile anesthetics bind to the same sites on albumin and, if so, whether they vary in their affinity for these sites. We show that three other fluorinated volatile anesthetics bind with varying affinity to fatty acid-displaceable domains on serum albumin: halothane, Kd = 1.3 +/- 0.2 mM; methoxyflurane, Kd = 2.6 +/- 0.3 mM; and sevoflurane, Kd = 4.5 +/- 0.6 mM. These three anesthetics inhibit isoflurane binding in a competitive manner: halothane, K(i) (inhibition constant) = 1.3 +/- 0.2 mM; methoxyflurane, K(i) = 2.5 +/- 0.4 mM; and sevoflurane, K(i) = 5.4 +/- 0.7 mM--similar to each anesthetic's respective Kd of binding to fatty acid displaceable sites. These results illustrate that a variety of volatile anesthetics can compete for binding to specific sites on a protein. PMID:8341659

Intercalation of XR5944 with the estrogen response element is modulated by the tri-nucleotide spacer sequence between half-sites

PubMed Central

Sidell, Neil; Mathad, Raveendra I.; Shu, Feng-jue; Zhang, Zhenjiang; Kallen, Caleb B.; Yang, Danzhou

2011-01-01

DNA-intercalating molecules can impair DNA replication, DNA repair, and gene transcription. We previously demonstrated that XR5944, a DNA bis-intercalator, specifically blocks binding of estrogen receptor-α (ERα) to the consensus estrogen response element (ERE). The consensus ERE sequence is AGGTCAnnnTGACCT, where nnn is known as the tri-nucleotide spacer. Recent work has shown that the tri-nucleotide spacer can modulate ERα-ERE binding affinity and ligand-mediated transcriptional responses. To further understand the mechanism by which XR5944 inhibits ERα-ERE binding, we tested its ability to interact with consensus EREs with variable tri-nucleotide spacer sequences and with natural but non-consensus ERE sequences using one dimensional nuclear magnetic resonance (1D 1H NMR) titration studies. We found that the tri-nucleotide spacer sequence significantly modulates the binding of XR5944 to EREs. Of the sequences that were tested, EREs with CGG and AGG spacers showed the best binding specificity with XR5944, while those spaced with TTT demonstrated the least specific binding. The binding stoichiometry of XR5944 with EREs was 2:1, which can explain why the spacer influences the drug-DNA interaction; each XR5944 spans four nucleotides (including portions of the spacer) when intercalating with DNA. To validate our NMR results, we conducted functional studies using reporter constructs containing consensus EREs with tri-nucleotide spacers CGG, CTG, and TTT. Results of reporter assays in MCF-7 cells indicated that XR5944 was significantly more potent in inhibiting the activity of CGG- than TTT-spaced EREs, consistent with our NMR results. Taken together, these findings predict that the anti-estrogenic effects of XR5944 will depend not only on ERE half-site composition but also on the tri-nucleotide spacer sequence of EREs located in the promoters of estrogen-responsive genes. PMID:21333738

Ganglioside inhibition of sup 125 I-plasmin binding to colorectal carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liepkalns, V.A.; Burtin, M.C.; Correc, P.

1990-01-01

The pre-incubation of human colorectal carcinoma cells SW 1116 with 25 to 100 uM purified gangliosides resulted in 35-60% inhibition of specific {sup 125}I-plasmin binding to the cell surface. After 5 to 6 days in culture, tumor cells were pre-incubated at 4 degrees for 1 to 4 h followed by post-incubation with {sup 125}I-plasmin by techniques previously described. At 25 uM the capacity for inhibition of plasmin binding was GT1b greater than GQ1b greater than or equal to GD1a greater than GM1 less than or equal to GgOse 4Cer. Thus a terminal sialyl moiety appears to be necessary (p lessmore » than 0.05) although exogenous N-acetyl neuraminic acid was ineffective (p greater than 0.05), indicating a role for the lipid portion of the ganglioside. Other (glyco)lipids such as sphingosine, fucolipid H-1 and sulfatide were without significant effect. The inhibition could not be reversed by the presence of 10 mM Ca+2, EDTA, pre-treatment of the cell with carboxypeptidase or pretreatment of plasmin with neuraminidases. The inhibition was however reversed by post-incubation in control medium without exogenous ganglioside. Cell counts determined prior to, and after ganglioside incubation showed that the effect was not due to cell death or detachment from the culture surface. The dissociation constant for {sup 125}I-plasmin binding was 5.6 x 10(-8) M (700,000 sites/cell), but in the presence of trisialoganglioside (GT1b), Scatchard plots suggested diversification of binding sites with 280,000 sites/cell at Kd 2.6 x 10(-8) M and 820,000 sites/cell at Kd 2.1 x 10(-7) M. Another interpretation of the Scatchard plot in the presence of ganglioside was that the glycolipid imposed negative cooperativity on plasmin binding to the cell surface. These results suggest that certain gangliosides can affect tumor cell invasiveness by altering protease binding to the cell surface.« less

Antibodies among men and children to placental-binding Plasmodium falciparum-infected erythrocytes that express var2csa.

PubMed

Beeson, James G; Ndungu, Francis; Persson, Kristina E M; Chesson, Joanne M; Kelly, Greg L; Uyoga, Sophie; Hallamore, Sandra L; Williams, Thomas N; Reeder, John C; Brown, Graham V; Marsh, Kevin

2007-07-01

During pregnancy, specific variants of Plasmodium falciparum-infected erythrocytes (IEs) can accumulate in the placenta through adhesion to chondroitin sulfate A (CSA) mediated by expression of PfEMP1 encoded by var2csa-type genes. Antibodies against these variants are associated with protection from maternal malaria. We evaluated antibodies among Kenyan, Papua New Guinean, and Malawian men and Kenyan children against two different CSA-binding P. falciparum isolates expressing var2csa variants. Specific IgG was present at significant levels among some men and children from each population, suggesting exposure to these variants is not exclusive to pregnancy. However, the level and prevalence of antibodies was substantially lower overall than exposed multigravidas. IgG-binding was specific and did not represent antibodies to subpopulations of non-CSA-binding IEs, and some sera inhibited IE adhesion to CSA. These findings have significant implications for understanding malaria pathogenesis and immunity and may be significant for understanding the acquisition of immunity to maternal malaria.

Protozoa lectins and their role in host-pathogen interactions.

PubMed

Singh, Ram Sarup; Walia, Amandeep Kaur; Kanwar, Jagat Rakesh

2016-01-01

Lectins are proteins/glycoproteins of non-immune origin that agglutinate red blood cells, lymphocytes, fibroblasts, etc., and bind reversibly to carbohydrates present on the apposing cells. They have at least two carbohydrate binding sites and their binding can be inhibited by one or more carbohydrates. Owing to carbohydrate binding specificity of lectins, they mediate cell-cell interactions and play role in protozoan adhesion and host cell cytotoxicity, thus are central to the pathogenic property of the parasite. Several parasitic protozoa possess lectins which mediate parasite adherence to host cells based on their carbohydrate specificities. These interactions could be exploited for development of novel therapeutics, targeting the adherence and thus helpful in eradicating wide spread of protozoan diseases. The current review highlights the present state knowledge with regard to protozoal lectins with an emphasis on their haemagglutination activity, carbohydrate specificity, characteristics and also their role in pathogenesis notably as adhesion molecules, thereby aiding the pathogen in disease establishment. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibition and Regulation of the Ergothioneine Biosynthetic Methyltransferase EgtD.

PubMed

Misson, Laëtitia; Burn, Reto; Vit, Allegra; Hildesheim, Julia; Beliaeva, Mariia A; Blankenfeldt, Wulf; Seebeck, Florian P

2018-05-18

Ergothioneine is an emerging factor in cellular redox homeostasis in bacteria, fungi, plants, and animals. Reports that ergothioneine biosynthesis may be important for the pathogenicity of bacteria and fungi raise the question as to how this pathway is regulated and whether the corresponding enzymes may be therapeutic targets. The first step in ergothioneine biosynthesis is catalyzed by the methyltransferase EgtD that converts histidine into N-α-trimethylhistidine. This report examines the kinetic, thermodynamic and structural basis for substrate, product, and inhibitor binding by EgtD from Mycobacterium smegmatis. This study reveals an unprecedented substrate binding mechanism and a fine-tuned affinity landscape as determinants for product specificity and product inhibition. Both properties are evolved features that optimize the function of EgtD in the context of cellular ergothioneine production. On the basis of these findings, we developed a series of simple histidine derivatives that inhibit methyltransferase activity at low micromolar concentrations. Crystal structures of inhibited complexes validate this structure- and mechanism-based design strategy.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J. H.; Cho, U. -S.; Karin, M.

Sestrins are highly conserved, stress-inducible proteins that inhibit target of rapamycin complex 1 (TORC1) signaling. After their transcriptional induction, both vertebrate and invertebrate Sestrins turn on the adenosine monophosphate (AMP)–activated protein kinase (AMPK), which activates the tuberous sclerosis complex (TSC), a key inhibitor of TORC1 activation. However, Sestrin overexpression, on occasion, can result in TORC1 inhibition even in AMPK-deficient cells. This effect has been attributed to Sestrin’s ability to bind the TORC1-regulating GATOR2 protein complex, which was postulated to control trafficking of TORC1 to lysosomes. How the binding of Sestrins to GATOR2 is regulated and how it contributes to TORC1more » inhibition are unknown. New findings suggest that the amino acid leucine specifically disrupts the association of Sestrin2 with GATOR2, thus explaining how leucine and related amino acids stimulate TORC1 activity. We discuss whether and how these findings fit what has already been learned about Sestrin-mediated TORC1 inhibition from genetic studies conducted in fruit flies and mammals.« less

Computational multiscale modeling in protein--ligand docking.

PubMed

Taufer, Michela; Armen, Roger; Chen, Jianhan; Teller, Patricia; Brooks, Charles

2009-01-01

In biological systems, the binding of small molecule ligands to proteins is a crucial process for almost every aspect of biochemistry and molecular biology. Enzymes are proteins that function by catalyzing specific biochemical reactions that convert reactants into products. Complex organisms are typically composed of cells in which thousands of enzymes participate in complex and interconnected biochemical pathways. Some enzymes serve as sequential steps in specific pathways (such as energy metabolism), while others function to regulate entire pathways and cellular functions [1]. Small molecule ligands can be designed to bind to a specific enzyme and inhibit the biochemical reaction. Inhibiting the activity of key enzymes may result in the entire biochemical pathways being turned on or off [2], [3]. Many small molecule drugs marketed today function in this generic way as enzyme inhibitors. If research identifies a specific enzyme as being crucial to the progress of disease, then this enzyme may be targeted with an inhibitor, which may slow down or reverse the progress of disease. In this way, enzymes are targeted from specific pathogens (e.g., virus, bacteria, fungi) for infectious diseases [4], [5], and human enzymes are targeted for noninfectious diseases such as cardiovascular disease, cancer, diabetes, and neurodegenerative diseases [6].

Triple helix-forming oligonucleotide corresponding to the polypyrimidine sequence in the rat alpha 1(I) collagen promoter specifically inhibits factor binding and transcription.

PubMed

Kovacs, A; Kandala, J C; Weber, K T; Guntaka, R V

1996-01-19

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of alpha 1(I) collagen promoter between nucleotides - 200 and -140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between -172 and -138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of alpha 1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.

Test/QA Plan for Verification of Microcystin Test Kits

EPA Science Inventory

Microcystin test kits are used to quantitatively measure total microcystin in recreational waters. These test kits are based on enzyme-linked immunosorbent assays (ELISA) with antibodies that bind specifically to microcystins or phosphate activity inhibition where the phosphatas...

Molecular insight of isotypes specific β-tubulin interaction of tubulin heterodimer with noscapinoids

NASA Astrophysics Data System (ADS)

Santoshi, Seneha; Naik, Pradeep K.

2014-07-01

Noscapine and its derivatives bind stoichiometrically to tubulin, alter its dynamic instability and thus effectively inhibit the cellular proliferation of a wide variety of cancer cells including many drug-resistant variants. The tubulin molecule is composed of α- and β-tubulin, which exist as various isotypes whose distribution and drug-binding properties are significantly different. Although the noscapinoids bind to a site overlapping with colchicine, their interaction is more biased towards β-tubulin. In fact, their precise interaction and binding affinity with specific isotypes of β-tubulin in the αβ-heterodimer has never been addressed. In this study, the binding affinity of a panel of noscapinoids with each type of tubulin was investigated computationally. We found that the binding score of a specific noscapinoid with each type of tubulin isotype is different. Specifically, amino-noscapine has the highest binding score of -6.4, -7.2, -7.4 and -7.3 kcal/mol with αβI, αβII, αβIII and αβIV isotypes, respectively. Similarly 10 showed higher binding affinity of -6.8 kcal/mol with αβV, whereas 8 had the highest binding affinity of -7.2, -7.1 and -7.2 kcal/mol, respectively with αβVI, αβVII and αβVIII isotypes. More importantly, both amino-noscapine and its clinical derivative, bromo-noscapine have the highest binding affinity of -46.2 and -38.1 kcal/mol against αβIII (overexpression of αβIII has been associated with resistance to a wide range of chemotherapeutic drugs for several human malignancies) as measured using MM-PBSA. Knowledge of the isotype specificity of the noscapinoids may allow for development of novel therapeutic agents based on this class of drugs.

Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication.

PubMed

Munke, Anna; Persson, Jonas; Weiffert, Tanja; De Genst, Erwin; Meisl, Georg; Arosio, Paolo; Carnerup, Anna; Dobson, Christopher M; Vendruscolo, Michele; Knowles, Tuomas P J; Linse, Sara

2017-06-20

The aggregation of the amyloid β peptide (Aβ) into amyloid fibrils is a defining characteristic of Alzheimer's disease. Because of the complexity of this aggregation process, effective therapeutic inhibitors will need to target the specific microscopic steps that lead to the production of neurotoxic species. We introduce a strategy for generating fibril-specific antibodies that selectively suppress fibril-dependent secondary nucleation of the 42-residue form of Aβ (Aβ42). We target this step because it has been shown to produce the majority of neurotoxic species during aggregation of Aβ42. Starting from large phage display libraries of single-chain antibody fragments (scFvs), the three-stage approach that we describe includes ( i ) selection of scFvs with high affinity for Aβ42 fibrils after removal of scFvs that bind Aβ42 in its monomeric form; ( ii ) ranking, by surface plasmon resonance affinity measurements, of the resulting candidate scFvs that bind to the Aβ42 fibrils; and ( iii ) kinetic screening and analysis to find the scFvs that inhibit selectively the fibril-catalyzed secondary nucleation process in Aβ42 aggregation. By applying this approach, we have identified four scFvs that inhibit specifically the fibril-dependent secondary nucleation process. Our method also makes it possible to discard antibodies that inhibit elongation, an important factor because the suppression of elongation does not target directly the production of toxic oligomers and may even lead to its increase. On the basis of our results, we suggest that the method described here could form the basis for rationally designed immunotherapy strategies to combat Alzheimer's and related neurodegenerative diseases.

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells

PubMed Central

Tahara, Atsuo; Tsukada, Junko; Tomura, Yuichi; Wada, Koh-ichi; Kusayama, Toshiyuki; Ishii, Noe; Yatsu, Takeyuki; Uchida, Wataru; Tanaka, Akihiro

2000-01-01

[3H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [3H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (Kd) of 0.76 nM and a maximum receptor density (Bmax) of 153 fmol mg−1 protein. The Hill coefficient (nH) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [3H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [3H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu1,6]-oxytocin>AVP= atosiban>d(CH2)5Tyr(Me)AVP>[Thr4,Gly7]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca2+]i increase and hyperplasia. In contrast, the V1A receptor selective antagonist, SR 49059, and the V2 receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca2+]i increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca2+]i increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [3H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca2+]i increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. PMID:10694212

Modulatory effects of heparin and short-length oligosaccharides of heparin on the metastasis and growth of LMD MDA-MB 231 breast cancer cells in vivo

PubMed Central

Mellor, P; Harvey, J R; Murphy, K J; Pye, D; O'Boyle, G; Lennard, T W J; Kirby, J A; Ali, S

2007-01-01

Expression of the chemokine receptor CXCR4 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express CXCL12. In this study, we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides. Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I125 CXCL12. Heparin dodecasaccharides were found to be the minimal chain length required to efficiently bind CXCL12 (71% inhibition; P<0.001). These oligosaccharides also significantly inhibited CXCL12-induced migration of CXCR4-expressing LMD MDA-MB 231 breast cancer cells. In addition, heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product, Tinzaparin. When given subcutaneously in a SCID mouse model of human breast cancer, heparin dodecasaccharides had no effect on the number of lung metastases, but did however inhibit (P<0.05) tumour growth (lesion area) compared to control groups. In contrast, polymeric heparin significantly inhibited both the number (P<0.001) and area of metastases, suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases. PMID:17726466

Partial purification of the mu opioid receptor irreversibly labeled with (/sup 3/H)b-funaltrexamine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu-Chen, L.Y.; Phillips, C.A.; Tam, S.W.

1986-03-01

The mu opioid receptor in bovine striatal membranes was specifically and irreversibly labeled by incubation with 5 nM (/sup 3/H)..beta..-funaltrexamine (approx.-FNA) at 37/sup 0/C for 90 min in the presence of 100 mM NaCl. The specific and irreversible binding of (/sup 3/H)..beta..-FNA as defined by that blocked by 1 /sup +/M naloxone was about 60% of total irreversible binding. The specific irreversible binding was saturable, stereospecific, time-, temperature, and tissue-dependent. Mu opioid ligands were much more potent than delta or kappa ligands in inhibiting the specific irreversible labeling. SDS polyacrylamide gel electrophoresis of solubilized membranes in the presence of 2-mercaptoethanolmore » yielded a major radiolabeled broad band of MW 68-97K daltons, characteristic of a glycoprotein band. This band was not observed in membranes labeled in the presence of excess unlabeled naloxone. The glycoprotein nature of the (/sup 3/H)..beta..-FNA-labeled opioid receptor was confirmed by its binding to a wheat germ agglutinin-Sepharose column and its elution with N-acetylglucosamine.« less

The hedgehog regulated oncogenes Gli1 and Gli2 block myoblast differentiation by inhibiting MyoD-mediated transcriptional activation

PubMed Central

Gerber, AN; Wilson, CW; Li, Y-J; Chuang, P-T

2012-01-01

The mechanism by which activation of the Hedgehog (Hh) pathway modulates differentiation and promotes oncogenesis in specific tissues is poorly understood. We therefore, analysed rhabdomyosarcomas from mice that were haploinsufficient for the Hh-binding protein, Hip1, or for the Hh receptor, Patched 1 (Ptch1). Transfection of the Hh-regulated transcription factor Gli1, which is expressed in a subset of mouse and human rhabdomyosarcomas, suppressed differentiation of myogenic rhabdomyosarcoma lines generated from Hip1+/− and Ptch1+/− mice. The closely related factor, Gli2, had similar effects. Gli1 and Gli2 inhibited myogenesis by repressing the capacity of MyoD to activate transcription. Deletion analysis of Gli1 indicated that multiple domains of Gli1 are required for efficient inhibition of MyoD. Gli1 reduced the ability of MyoD to heterodimerize with E12 and bind DNA, providing one mechanism whereby the Gli proteins modulate the activity of MyoD. This novel activity of Gli proteins provides new insights into how Hh signaling modulates terminal differentiation through inhibition of tissue-specific factors such as MyoD. This mechanism may contribute to the broad role of Hh signaling and the Gli proteins in differentiation decisions and cancer formation. PMID:16964293

Monitoring DNA triplex formation using multicolor fluorescence and application to insulin-like growth factor I promoter downregulation.

PubMed

Hégarat, Nadia; Novopashina, Darya; Fokina, Alesya A; Boutorine, Alexandre S; Venyaminova, Alya G; Praseuth, Danièle; François, Jean-Christophe

2014-03-01

Inhibition of insulin-like growth factor I (IGF-I) signaling is a promising antitumor strategy and nucleic acid-based approaches have been investigated to target genes in the pathway. Here, we sought to modulate IGF-I transcriptional activity using triple helix formation. The IGF-I P1 promoter contains a purine/pyrimidine (R/Y) sequence that is pivotal for transcription as determined by deletion analysis and can be targeted with a triplex-forming oligonucleotide (TFO). We designed modified purine- and pyrimidine-rich TFOs to bind to the R/Y sequence. To monitor TFO binding, we developed a fluorescence-based gel-retardation assay that allowed independent detection of each strand in three-stranded complexes using end-labeling with Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterized TFOs for their ability to inhibit restriction enzyme activity, compete with DNA-binding proteins and inhibit IGF-I transcription in reporter assays. TFOs containing modified nucleobases, 5-methyl-2'-deoxycytidine and 5-propynyl-2'-deoxyuridine, specifically inhibited restriction enzyme cleavage and formed triplexes on the P1 promoter fragment. In cells, deletion of the R/Y-rich sequence led to 48% transcriptional inhibition of a reporter gene. Transfection with TFOs inhibited reporter gene activity to a similar extent, whereas transcription from a mutant construct with an interrupted R/Y region was unaffected, strongly suggesting the involvement of triplex formation in the inhibitory mechanisms. Our results indicate that nuclease-resistant TFOs will likely inhibit endogenous IGF-I gene function in cells. © 2014 FEBS.

Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morris, D.I.; Robbins, J.D.; Ruoho, A.E.

1991-07-15

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of (3H)forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited (3H)forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP {gamma} S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolinmore » but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP {gamma} S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.« less

FMRFamide: low affinity inhibition of opioid binding to rabbit brain membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, X.Z.; Raffa, R.B.

1986-03-05

FMRFamide (Phe-Met-Arg-Phe-NH/sub 2/) was first isolated from the ganglia of molluscs by Price and Greenberg in 1977. The peptide was subsequently shown to have diverse actions on various types of molluscan and mammalian tissues. The presence of immunoreactive FMRFamide-like material (irFMRF) in multiple areas of rat brain, spinal cord, and gastrointestinal tract suggests that irFMRF may have a physiological role in mammals. Tang, Yang and Costa recently demonstrated that FMRFamide attenuates morphine antinociception in rats and postulated, based on this and several other lines of evidence, that irFMRF might be an endogenous opioid antagonist. In the present study, they testedmore » the ability of FMRFamide to inhibit the binding of opioid receptor ligands to rabbit membrane preparations. FMRFamide inhibited the specific binding of both /sup 3/(H)-dihydromorphine and /sup 3/(H)-ethylketocyclazocine (IC/sub 50/ = 14 ..mu..M and 320 ..mu..M, respectively) in a dose-related manner, suggesting that FMRFamide may affect binding to at least two types of opioid receptors (mu and kappa). These data are consistent with the concept that irFMRF might act as an endogenous opioid antagonist. However, the low affinity of FMRFamide leaves open the possibility of another mechanism of opioid antagonism, such as neuromodulation.« less

A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC.

PubMed

Kenesi, Erzsébet; Carbonell, Alberto; Lózsa, Rita; Vértessy, Beáta; Lakatos, Lóránt

2017-07-27

In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Novel cyclo-peptides inhibit Ebola pseudotyped virus entry by targeting primed GP protein.

PubMed

Li, Quanjie; Ma, Ling; Yi, Dongrong; Wang, Han; Wang, Jing; Zhang, Yongxin; Guo, Ying; Li, Xiaoyu; Zhou, Jinming; Shi, Yi; Gao, George F; Cen, Shan

2018-07-01

Ebola virus (EBOV) causes fatal hemorrhagic fever with high death rates in human. Currently, there are no available clinically-approved prophylactic or therapeutic treatments. The recently solved crystal structure of cleavage-primed EBOV glycoprotein (GPcl) in complex with the C domain of endosomal protein Niemann-Pick C1 (NPC1) provides a new target for the development of EBOV entry inhibitors. In this work, a computational approach using docking and molecular dynamic simulations is carried out for the rational design of peptide inhibitors. A novel cyclo-peptide (Pep-3.3) was identified to target at the late stage of EBOV entry and exhibit specific inhibitory activity against EBOV-GP pseudotyped viruses, with 50% inhibitory concentration (IC50) of 5.1 μM. In vitro binding assay and molecular simulations revealed that Pep-3.3 binds to GPcl with a KD value of 69.7 μM, through interacting with predicted residues in the hydrophobic binding pocket of GPcl. Mutation of predicted residues T83 caused resistance to Pep-3.3 inhibition in viral infectivity, providing preliminary support for the model of the peptide binding to GPcl. This study demonstrates the feasibility of inhibiting EBOV entry by targeting GPcl with peptides. Copyright © 2018 Elsevier B.V. All rights reserved.

Binding of KATP channel modulators in rat cardiac membranes

PubMed Central

Löffler-Walz, Cornelia; Quast, Ulrich

1998-01-01

The binding of [3H]-P1075, a potent opener of adenosine-5′-triphosphate-(ATP)-sensitive K+ channels, was studied in a crude heart membrane preparation of the rat, at 37°C.Binding required MgATP. In the presence of an ATP-regenerating system, MgATP supported [3H]-P1075 binding with an EC50 value of 100 μM and a Hill coefficient of 1.4.In saturation experiments [3H]-P1075 binding was homogeneous with a KD value of 6±1 nM and a binding capacity (Bmax) of 33±3 fmol mg−1 protein.Upon addition of an excess of unlabelled P1075, the [3H]-P1075-receptor complex dissociated in a mono-exponential manner with a dissociation rate constant of 0.13±0.01 min−1. If a bi-molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 nM−1 min−1. From the kinetic experiments the KD value was calculated as 4.7±0.6 nM.Openers of the ATP-sensitive K+ channel belonging to different structural classes inhibited specific [3H]-P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta.Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited [3H]-P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC50 value of glibenclamide being ≈amp;90 nM; affinities for the low affinity component were in the μM concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC50 value of 6 μM, a maximum inhibition of 94% and a Hill coefficient of 1.5.It is concluded that binding studies with [3H]-P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP-regenerating system. The pharmacological profile of the [3H]-P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR2A) and vascular smooth muscle cells (SUR2B), differs from that expected for SUR2A and SUR2B. PMID:9579735

Neuromedin B receptor in esophagus: evidence for subtypes of bombesin receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Von Schrenck, T.; Heinz-Erian, P.; Moran, T.

1989-04-01

To identify receptors for bombesin-related peptides in the rat esophagus, we measured binding of 125I-Bolton-Hunter neuromedin B (125I-BH-neuromedin B) and 125I-(Tyr4)bombesin to tissue sections from the rat esophagus and compared the results with those for rat pancreas. Esophagus bound both tracers, whereas pancreas bound only 125I-(Tyr4)bombesin. In each tissue binding was saturable, dependent on pH, on time, and on temperature, reversible, and specific. Autoradiography demonstrated binding of both tracers only to the muscularis mucosae of the esophagus and binding of 125I-(Tyr4)bombesin diffusely over pancreatic acini. In the esophagus, the relative potencies for inhibition of binding of both tracers were asmore » follows: neuromedin B greater than bombesin greater than GRP = neuromedin C; similar relative potencies were found for causing contraction of muscle strips from whole esophagus and from the isolated muscularis mucosae. In pancreas tissue sections and dispersed acini, the relative potencies for inhibition of binding of 125I-(Tyr4)bombesin were as follows: bombesin greater than GRP = neuromedin C much greater than neuromedin B. Similar relative potencies were found for stimulation of enzyme secretion from dispersed pancreatic acini. Computer analysis in both tissues demonstrated only a single binding site. The present study demonstrates that rat esophagus muscle possesses specific receptors for bombesin-related peptides. Furthermore, this study shows that the esophageal bombesin receptors represent a previously unidentified class of bombesin receptors in that they have a higher affinity for neuromedin B than for bombesin. In contrast, the pancreatic bombesin receptors have, like all other bombesin receptors described to date, a high affinity for bombesin, but low affinity for neuromedin B.« less

Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region

PubMed Central

Essono, Sosthène; Mondielli, Grégoire; Lamourette, Patricia; Boquet, Didier; Grassi, Jacques; Marchot, Pascale

2013-01-01

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis. PMID:24146971

Locating herpesvirus Bcl-2 homologs in the specificity landscape of anti-apoptotic Bcl-2 proteins

PubMed Central

Foight, Glenna Wink; Keating, Amy E.

2015-01-01

Viral homologs of the anti-apoptotic Bcl-2 proteins are highly diverged from their mammalian counterparts, yet they perform overlapping functions by binding and inhibiting BH3 motif-containing proteins. We investigated the BH3 binding properties of the herpesvirus Bcl-2 homologs KSBcl-2, BHRF1, and M11, as they relate to those of the human Bcl-2 homologs Mcl-1, Bfl-1, Bcl-w, Bcl-xL, and Bcl-2. Analysis of the sequence and structure of the BH3 binding grooves showed that, despite low sequence identity, M11 has structural similarities to Bcl-xL, Bcl-2, and Bcl-w. BHRF1 and KSBcl-2 are more structurally similar to Mcl-1 than to the other human proteins. Binding to human BH3-like peptides showed that KSBcl-2 has similar specificity to Mcl-1, and BHRF1 has a restricted binding profile; M11 binding preferences are distinct from those of Bcl-xL, Bcl-2 and Bcl-w. Because KSBcl-2 and BHRF1 are from human herpesviruses associated with malignancies, we screened computationally designed BH3 peptide libraries using bacterial surface display to identify selective binders of KSBcl-2 or BHRF1. The resulting peptides bound to KSBcl-2 and BHRF1 in preference to Bfl-1, Bcl-w, Bcl-xL, and Bcl-2, but showed only modest specificity over Mcl-1. Rational mutagenesis increased specificity against Mcl-1, resulting in a peptide with a dissociation constant of 2.9 nM for binding to KSBcl-2 and >1000-fold specificity over human Bcl-2 proteins, and a peptide with >70-fold specificity for BHRF1. In addition to providing new insights into viral Bcl-2 binding specificity, this study will inform future work analyzing the interaction properties of homologous binding domains and designing specific protein interaction partners. PMID:26009469

Effects of a Variety of Food Extracts and Juices on the Specific Binding Ability of Norovirus GII.4 P Particles

PubMed Central

LI, DAN; BAERT, LEEN; XIA, MING; ZHONG, WEIMING; JIANG, XI; UYTTENDAELE, MIEKE

2014-01-01

The effects of 13 food extracts and juices, including shellfish, fruits, and vegetables, on the binding ability of human norovirus (NoV) were examined, using P particles of human NoV GII.4 as a research surrogate. The enhancements (positive values) or reductions (negative values) of NoV P particle detection (changes in optical density at 450 nm) in the presence of different food extracts and juices as compared with P particles diluted in phosphate-buffered saline were tested by saliva-binding, enzyme-linked immunosorbent assay in triplicate. In the presence of different food extracts and juices at different concentrations, an increase or decrease of the receptor-binding ability of the NoV P particles was observed. Due to a higher specific binding and thus a higher accumulation of the viral particles, oysters may be contaminated with human NoV more often than other shellfish species (mussel, hard clams, and razor clams). Cranberry and pomegranate juices were shown to reduce the specific binding ability of human NoV P particles. No such binding inhibition effects were observed for the other tested extracts of fresh produce (strawberry, blackberry, blueberry, cherry tomato, spinach, romaine lettuce) or, notably, for raspberry, which has been associated with human NoV outbreaks. PMID:22980024

Effects of a variety of food extracts and juices on the specific binding ability of norovirus GII.4 P particles.

PubMed

Li, Dan; Baert, Leen; Xia, Ming; Zhong, Weiming; Jiang, Xi; Uyttendaele, Mieke

2012-07-01

The effects of 13 food extracts and juices, including shellfish, fruits, and vegetables, on the binding ability of human norovirus (NoV) were examined, using P particles of human NoV GII.4 as a research surrogate. The enhancements (positive values) or reductions (negative values) of NoV P particle detection (changes in optical density at 450 nm) in the presence of different food extracts and juices as compared with P particles diluted in phosphate-buffered saline were tested by saliva-binding, enzyme-linked immunosorbent assay in triplicate. In the presence of different food extracts and juices at different concentrations, an increase or decrease of the receptor-binding ability of the NoV P particles was observed. Due to a higher specific binding and thus a higher accumulation of the viral particles, oysters may be contaminated with human NoV more often than other shellfish species (mussel, hard clams, and razor clams). Cranberry and pomegranate juices were shown to reduce the specific binding ability of human NoV P particles. No such binding inhibition effects were observed for the other tested extracts of fresh produce (strawberry, blackberry, blueberry, cherry tomato, spinach, romaine lettuce) or, notably, for raspberry, which has been associated with human NoV outbreaks.

Developing recombinant HPA-1a-specific antibodies with abrogated Fcgamma receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia.

PubMed

Ghevaert, Cedric; Wilcox, David A; Fang, Juan; Armour, Kathryn L; Clark, Mike R; Ouwehand, Willem H; Williamson, Lorna M

2008-08-01

Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal generation of antibodies specific for paternal platelet antigens and can lead to fetal intracranial hemorrhage. A SNP in the gene encoding integrin beta3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a-specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a-specific scFv (B2) with an IgG1 constant region modified to minimize Fcgamma receptor-dependent platelet destruction (G1Deltanab). B2G1Deltanab saturated HPA-1a+ platelets and substantially inhibited binding of clinical HPA-1a-specific sera to HPA-1a+ platelets. The response of monocytes to B2G1Deltanab-sensitized platelets was substantially less than their response to unmodified B2G1, as measured by chemiluminescence. In addition, B2G1Deltanab inhibited chemiluminescence induced by B2G1 and HPA-1a-specific sera. In a chimeric mouse model, B2G1 and polyclonal Ig preparations from clinical HPA-1a-specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia. As the Deltanab constant region is uninformative in mice, F(ab')2 B2G1 was used as a proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a-specific antibodies. These results provide rationale for human clinical studies.

Uptake of Clostridium botulinum C3 Exoenzyme into Intact HT22 and J774A.1 Cells

PubMed Central

Rohrbeck, Astrid; von Elsner, Leonie; Hagemann, Sandra; Just, Ingo

2015-01-01

The Clostridium botulinum C3 exoenzyme selectively ADP-ribosylates low molecular weight GTP-binding proteins RhoA, B and C. This covalent modification inhibits Rho signaling activity, resulting in distinct actin cytoskeleton changes. Although C3 exoenzyme has no binding, the translocation domain assures that C3 enters cells and acts intracellularly. C3 uptake is thought to occur due to the high concentration of the C3 enzyme. However, recent work indicates that C3 is selectively endocytosed, suggesting a specific endocytotic pathway, which is not yet understood. In this study, we show that the C3 exoenzyme binds to cell surfaces and is internalized in a time-dependent manner. We show that the intermediate filament, vimentin, is involved in C3 uptake, as indicated by the inhibition of C3 internalization by acrylamide, a known vimentin disruption agent. Inhibition of C3 internalization was not observed by chemical inhibitors, like bafilomycin A, methyl-β-cyclodextrin, nocodazole or latrunculin B. Furthermore, the internalization of C3 exoenzyme was markedly inhibited in dynasore-treated HT22 cells. Our results indicate that C3 internalization depends on vimentin and does not depend strictly on both clathrin and caveolae. PMID:25648844

Curcumin directly inhibits the transport activity of GLUT1

PubMed Central

Gunnink, Leesha K.; Alabi, Ola D.; Kuiper, Benjamin D.; Gunnink, Stephen M.; Schuiteman, Sam J.; Strohbehn, Lauren E.; Hamilton, Kathryn E.; Wrobel, Kathryn E.; Louters, Larry L.

2016-01-01

Curcumin, a major ingredient in turmeric, has a long history of medicinal applications in a wide array of maladies including treatment for diabetes and cancer. Seemingly counterintuitive to the documented hypoglycemic effects of curcumin, however, a recent report indicates that curcumin directly inhibits glucose uptake in adipocytes. The major glucose transporter in adipocytes is GLUT4. Therefore, this study investigates the effects of curcumin in cell lines where the major transporter is GLUT1. We report that curcumin has an immediate inhibitory effect on basal glucose uptake in L929 fibroblast cells with a maximum inhibition of 80% achieved at 75 μM curcumin. Curcumin also blocks activation of glucose uptake by azide, glucose deprivation, hydroxylamine, or phenylarsine oxide. Inhibition does not increase with exposure time and the inhibitory effects reverse within an hour. Inhibition does not appear to involve a reaction between curcumin and the thiol side chain of a cysteine residue since neither prior treatment of cells with iodoacetamide nor curcumin with cysteine alters curcumin’s inhibitory effects. Curcumin is a mixed inhibitor reducing the Vmax of 2DG transport by about half with little effect on the Km. The inhibitory effects of curcumin are not additive to the effects of cytochalasin B and 75 μM curcumin actually reduces specific cytochalasin B binding by 80%. Taken together, the data suggest that curcumin binds directly to GLUT1 at a site that overlaps with the cytochalasin B binding site and thereby inhibits glucose transport. A direct inhibition of GLUT proteins in intestinal epithelial cells would likely reduce absorption of dietary glucose and contribute to a hypoglycemic effect of curcumin. Also, inhibition of GLUT1 activity might compromise cancer cells that overexpress GLUT1 and be another possible mechanism for the documented anticancer effects of curcumin. PMID:27039889

Neuron-Glia Adhesion is Inhibited by Antibodies to Neural Determinants

NASA Astrophysics Data System (ADS)

Grumet, M.; Rutishauser, U.; Edelman, G. M.

1983-10-01

Suspensions of embryonic chick neuronal cells adhered to monolayers of glial cells, but few neurons bound to control monolayers of fibroblastic cells from meninges or skin. Neuronal cell-glial cell adhesion was inhibited by prior incubation of the neurons with Fab' fragments of antibodies to neuronal membranes. In contrast, antibodies to the neural cell adhesion molecule (N-CAM) did not inhibit the binding. These results suggest that a specific adhesive mechanism between neurons and glial cells exists and that it is mediated by CAM's that differ from those so far identified.

Adenine nucleotide transport in sonic submitochondrial particles. Kinetic properties and binding of specific inhibitors.

PubMed

Lauquin, G J; Villiers, C; Michejda, J W; Hryniewiecka, L V; Vignais, P V

1977-05-11

1. A procedure for preparation of sonic submitochondrial particles competent for adenine nucleotide transport is described. ADP or ATP transport was assayed, in the presence of oligomycin, in a saline medium made of 0.125 M KCl, 1 mM EDTA, 10 mM 4-morpholinopropane sulfonic acid buffer, pH 6.5. 2. Sonic particles transport ADP and ATP by an exchange diffusion process. Externally added ADP (or ATP) is exchanged with internal ADP and ATP with a stoichiometry of one to one. The V value for ADP transport 5 degrees C was between 2 and 3 nmol/min per mg protein. 3. The transport system in sonic particles is specific for ADP and ATP. It is strongly dependent on temperature. The activation energy between 0 and 9 degrees C is approx. 35 kcal/mol. The optimum pH is 6.5, 4, Like in intact mitochondria, externally added ADP is transported into sonic particles faster at a given concentration than externally added ATP. The V value for ADP transport is 1.5-2 times higher than the V value for ATP transport. 5. The transition from the energized to the deenergized state in sonic particles results in a decrease of the pH gradient across the membrane (internal pH less than external pH) and in a 2-4 fold increase in the Km value for ATP. This latter effect is opposite that found for transport of added ATP in intact mitochondria (Souverijn, J.H.M., Huisman, L.A., Rosing J. and Kemp, Jr., A. (1973) Biochim. Biophys. Acta 305, 185-198). Energization has no effect on the V value of ATP transport in sonic particles. 6. In contrast to intact mitochondria, inhibition of ADP transport in sonic particles by bongkrekic acid does not have any lag-time and does not depend on pH. The inhibition caused by bongkrekic acid is a mixed type inhibition with a Ki value of 1.2 micronM. Atractyloside and carboxyatractyloside do not inhibit ADP transport in sonic particles, unless the particles have been preloaded with these inhibitors during the sonication. 7. Palmityl-CoA added to sonic particles inhibits efficiently ADP transport. The mixed type inhibition found with palmityl-CoA has a Ki value of 1.6 micronM. 8. [3H]Bongkrekic acid binds to sonic particles readily and with high affinity. Bongkrekic acic binding to sonic particles does not depend on pH and it has a saturation plateau, corresponding approximately to 1.3 mol of site per mol of cytochrome a. The number of [3H]atracytloside binding sites is much lower (one-fifth of the bongkrekic acid). External carboxyatractyloside does not compete with [3H]bongkrekic acid for binding to sonic particles. However, when carboxyatractyloside is present inside the particles, it inhibits the binding of [3H]bongkrekic acid.

Evidence that forskolin binds to the glucose transporter of human erythrocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lavis, V.R.; Lee, D.P.; Shenolikar, S.

1987-10-25

Binding of (4-/sup 3/H)cytochalasin B and (12-/sup 3/H)forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of (12-/sup 3/H)forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of (12-/sup 3/H)forskolin and (4-/sup 3/H)cytochalasin B equivalently, with relative potencies parallel to their reported affinitiesmore » for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of (4-/sup 3/H)cytochalasin B or (12-/sup 3/H)forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either (4-/sup 3/H)cytochalasin B or (12-/sup 3/H)forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.« less

Method for screening inhibitors of the toxicity of Bacillus anthracis

DOEpatents

Cirino, Nick M.; Jackson, Paul J.; Lehnert, Bruce E.

2001-01-01

The protective antigen (PA) of Bacillus anthracis is integral to the mechanism of anthrax poisoning. The cloning, expression and purification of a 32 kDa B. anthracis PA fragment (PA32) is described. This fragment has also been expressed as a fusion construct to stabilized green fluorescent protein (EGFP-PA32). Both proteins were capable of binding to specific cell surface receptors as determined by fluorescent microscopy and a flow cytometric assay. To confirm binding specificity in the flow cytometric assay, non-fluorescent PA83 or PA32 was used to competitively inhibit fluorescent EGFP-PA32 binding to cell receptors. This assay can be employed as a rapid screen for compounds which disrupts binding of PA to cells. Additionally, the high intracellular expression levels and ease of purification make this recombinant protein an attractive vaccine candidate or therapeutic treatment for anthrax poisoning.

Binding of [35S]saccharin to a protein fraction of rat tongue epithelia.

PubMed

Shimazaki, K; Sato, M; Takegami, T

1981-11-05

The binding of [35S]saccharin to ammonium sulfate fractions from homogenates of rat tongue epithelia was measured by equilibrium dialysis. The 40--60% saturated ammonium sulfate fraction from the buffer-soluble fraction had the highest saccharin-binding activity. Binding of [35S]saccharin to the 40--60% ammonium sulfate fraction was inhibited by unlabeled saccharin sodium salt. The inhibition increased with increasing unlabeled saccharin concentration and was nearly complete above 10 mM. [35S]Saccharin binding to the 40--60% ammonium sulfate fraction extracted from the tongue epithelia was inhibited by glucose, lactose and sucrose, while binding to similar fractions from tongue muscle was not affected by these sugars. The inhibition of binding of labeled saccharin to the epithelial fraction increased with increasing glucose concentrations. About 35% of the binding was inhibited by 1 M glucose. No significant difference in the amount of inhibition was seen among the three sugars at 0.1 M. The 40--60% ammonium sulfate fraction from tongue epithelium devoid of taste buds bound much less [35S]saccharin than did a similar fraction from epithelium with taste buds. Binding of [35S]saccharin by the preparation from epithelium devoid of taste buds was not inhibited by glucose. The results provide evidence that the 40--60% ammonium sulfate fraction from tongue epithelia with taste buds contains a protein which binds saccharin and sugars. We hypothesize that it is a sweet taste receptor protein.

Nature of immobilization surface affects antibody specificity to placental alkaline phosphatase.

PubMed

Kumar, Mukesh; Khan, Imran; Sinha, Subrata

2015-01-01

Retention of native conformation of immobilized protein is essential for various applications including selection and detection of specific recombinant antibodies (scFvs). Placental alkaline phosphatase (PAP), an onco-fetal antigen expressed on the surface of several tumors, was immobilized on supermagnetic particles for selection of recombinant antibodies from a human phage display antibody library. The isolated antibodies were found to be cross-reactive to either of the isozymes of alkaline phosphatase, i.e., bone alkaline phosphatase (BAP) or intestinal alkaline phosphatase (IAP) and could not be used for tumor targeting. A specific anti-PAP monoclonal antibody H17E2 was tested for retention of specificity under these conditions. Binding of the antibody to magnetic beads conjugated IAP and BAP along with PAP and the ability of the two isozymes to inhibit its binding to PAP depicted the loss of isozyme specificity of the antibody. However, the antibody retained its specificity to PAP immobilized on polyvinyl chloride (PVC) surface. Enzyme activity was observed on both surfaces. This demonstrates that nature of immobilization may affect antigen-antibody binding in subtle ways, resulting in alteration of conformation of the epitopes. This may have consequences for determining the specificity of antibody binding for proteins that share a high degree of homology.

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy.

PubMed

Poiroux, Guillaume; Barre, Annick; van Damme, Els J M; Benoist, Hervé; Rougé, Pierre

2017-06-09

Aberrant O -glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O -glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola , and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O -glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors.

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy

PubMed Central

Poiroux, Guillaume; Barre, Annick; van Damme, Els J. M.; Benoist, Hervé; Rougé, Pierre

2017-01-01

Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors. PMID:28598369

Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A

PubMed Central

Kaminishi, Tatsuya; Schedlbauer, Andreas; Fabbretti, Attilio; Brandi, Letizia; Ochoa-Lizarralde, Borja; He, Cheng-Guang; Milón, Pohl; Connell, Sean R.; Gualerzi, Claudio O.; Fucini, Paola

2015-01-01

Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC. PMID:26464437

Effects of guanyl nucleotides on CCKB receptor binding in brain tissue and continuous cell lines: a comparative study.

PubMed

Kaufmann, R; Schöneberg, T; Henklein, P; Meyer, R; Martin, H; Ott, T

1995-07-01

The effects of non-hydrolyzable guanyl nucleotide analogue GTP-gamma S on CCKB receptor binding in human and guinea-pig cortex, Jurkat T-cells, rat pituitary GH3 cells, rat glioma C6 cells and human small cell lung cancer NCI-H69 cells were investigated by using [3H]CCK-8S saturation and competition binding studies. GTP-gamma S caused inhibition of specific [3H]CCK-8S binding in a concentration dependent manner with a plateau at 10-25 microM. 25 microM GTP-gamma S resulted in a small but significant increase in Kd and IC50 values with amount very similar in all CCKB receptor models tested. However, the maximal number of specific [3H]CCK-8S binding sites (Bmax) was unaffected. Results suggest that CCKB receptors are G-protein coupled in a similar way to human and guinea-pig cortex, Jurkat cells, GH3 cells, C6 cells and NCI-H69 cells.

Amiloride inhibits the initiation of Coxsackievirus and poliovirus RNA replication by inhibiting VPg uridylylation.

PubMed

Ogram, Sushma A; Boone, Christopher D; McKenna, Robert; Flanegan, James B

2014-09-01

The mechanism of amiloride inhibition of Coxsackievirus B3 (CVB3) and poliovirus type 1 (PV1) RNA replication was investigated using membrane-associated RNA replication complexes. Amiloride was shown to inhibit viral RNA replication and VPgpUpU synthesis. However, the drug had no effect on polymerase elongation activity during either (-) strand or (+) strand synthesis. These findings indicated that amiloride inhibited the initiation of RNA synthesis by inhibiting VPg uridylylation. In addition, in silico binding studies showed that amiloride docks in the VPg binding site on the back of the viral RNA polymerase, 3D(pol). Since VPg binding at this site on PV1 3D(pol) was previously shown to be required for VPg uridylylation, our results suggest that amiloride inhibits VPg binding to 3D(pol). In summary, our findings are consistent with a model in which amiloride inhibits VPgpUpU synthesis and viral RNA replication by competing with VPg for binding to 3D(pol). Copyright © 2014 Elsevier Inc. All rights reserved.

Resveratrol Directly Targets COX-2 to Inhibit Carcinogenesis

PubMed Central

Zykova, Tatyana A.; Zhu, Feng; Zhai, Xiuhong; Ma, Wei-ya; Ermakova, Svetlana P.; Lee, Ki Won; Bode, Ann M.; Dong, Zigang

2008-01-01

Targeted molecular cancer therapies can potentially deliver treatment directly to a specific protein or gene to optimize efficacy and reduce adverse side effects often associated with traditional chemotherapy. Key oncoprotein and oncogene targets are rapidly being identified based on their expression, pathogenesis and clinical outcome. One such protein target is cyclooxygenase-2 (COX-2), which is highly expressed in various cancers. Research findings suggest that resveratrol (3,5,4'-trihydroxy-trans-stilbene) demonstrates non-selective COX-2 inhibition. We report herein that resveratrol (RSVL) directly binds with COX-2 and this binding is absolutely required for RSVL's inhibition of the ability of human colon adenocarcinoma HT-29 cells to form colonies in soft agar. Binding of COX-2 with RSVL was compared with two RSVL analogues, 3,3’,4’,5’5’-pentahydroxy-trans-stilbene (RSVL-2) or 3,4’,5-trimethoxy-trans-stilbene (RSVL-3). The results indicated that COX-2 binds with RSVL-2 more strongly than with RSVL, but does not bind with RSVL-3. RSVL or RSVL-2, but not RSVL-3, inhibited COX-2-mediated PGE2 production in vitro and ex vivo. HT-29 human colon adenocarcinoma cells express high levels of COX-2 and either RSVL or RSVL-2, but not RSVL-3, suppressed anchorage independent growth of these cells in soft agar. RSVL or RSVL-2 (not RSVL-3) suppressed growth of COX-2+/+ cells by 60 or 80%, respectively. Notably, cells deficient in COX-2 were unresponsive to RSVL or RSVL-2. These data suggest that the anticancer effects of RSVL or RSLV-2 might be mediated directly through COX-2. PMID:18381589

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

PubMed Central

Takahashi, Yuji; Bigler, Dora; Ito, Yasuhiko; White, Judith M.

2001-01-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions. PMID:11294888

Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of beta1 integrin-associated proteins CD9, CD81, and CD98.

PubMed

Takahashi, Y; Bigler, D; Ito, Y; White, J M

2001-04-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-alpha6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-alpha6 mAb, or by mAbs against either the alphav or beta3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other beta1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg beta1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface "tetraspan web" facilitates fertilization and that it may do so by fostering ADAM-integrin interactions.

VKORC1 and VKORC1L1 have distinctly different oral anticoagulant dose-response characteristics and binding sites

PubMed Central

Czogalla, Katrin J.; Liphardt, Kerstin; Höning, Klara; Hornung, Veit; Biswas, Arijit; Watzka, Matthias

2018-01-01

Vitamin K reduction is catalyzed by 2 enzymes in vitro: the vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) and its isozyme VKORC1-like1 (VKORC1L1). In vivo, VKORC1 reduces vitamin K to sustain γ-carboxylation of vitamin K-dependent proteins, including coagulation factors. Inhibition of VKORC1 by oral anticoagulants (OACs) is clinically used in therapy and in prevention of thrombosis. However, OACs also inhibit VKORC1L1, which was previously shown to play a role in intracellular redox homeostasis in vitro. Here, we report data for the first time on specific inhibition of both VKOR enzymes for various OACs and rodenticides examined in a cell-based assay. Effects on endogenous VKORC1 and VKORC1L1 were independently investigated in genetically engineered HEK 293T cells that were knocked out for the respective genes by CRISPR/Cas9 technology. In general, dose-responses for 4-hydroxycoumarins and 1,3-indandiones were enzyme-dependent, with lower susceptibility for VKORC1L1 compared with VKORC1. In contrast, rodenticides exhibited nearly identical dose-responses for both enzymes. To explain the distinct inhibition pattern, we performed in silico modeling suggesting different warfarin binding sites for VKORC1 and VKORC1L1. We identified arginine residues at positions 38, 42, and 68 in the endoplasmatic reticulum luminal loop of VKORC1L1 responsible for charge-stabilized warfarin binding, resulting in a binding pocket that is diametrically opposite to that of VKORC1. In conclusion, our findings provide insight into structural and molecular drug binding on VKORC1, and especially on VKORC1L1. PMID:29581108

Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs

PubMed Central

García-Chapa, Meritxell; López-Moya, Juan José; Burgyán, József

2010-01-01

RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC. PMID:20657820

Increased actin polymerization reduces the inhibition of serum response factor activity by Yin Yang 1.

PubMed Central

Ellis, Peter D; Martin, Karen M; Rickman, Colin; Metcalfe, James C; Kemp, Paul R

2002-01-01

Recent evidence has implicated CC(A/T(richG))GG (CArG) boxes, binding sites for serum response factor (SRF), in the regulation of expression of a number of genes in response to changes in the actin cytoskeleton. In many cases, the activity of SRF at CArG boxes is modulated by transcription factors binding to overlapping (e.g. Yin Yang 1, YY1) or adjacent (e.g. ets) binding sites. However, the mechanisms by which SRF activity is regulated by the cytoskeleton have not been determined. To investigate these mechanisms, we screened for cells that did or did not increase the activity of a fragment of the promoter for a smooth-muscle (SM)-specific gene SM22alpha, in response to changes in actin cytoskeletal polymerization induced by LIM kinase. These experiments showed that vascular SM cells (VSMCs) and C2C12 cells increased the activity of promoters containing at least one of the SM22alpha CArG boxes (CArG near) in response to LIM kinase, whereas P19 cells did not. Bandshift assays using a probe to CArG near showed that P19 cells lacked detectable YY1 DNA binding to the CArG box in contrast with the other two cell types. Expression of YY1 in P19 cells inhibited SM22alpha promoter activity and conferred responsiveness to LIM kinase. Mutation of the CArG box to inhibit YY1 or SRF binding indicated that both factors were required for the LIM kinase response in VSMCs and C2C12 cells. The data indicate that changes in the actin cytoskeletal organization modify SRF activity at CArG boxes by modulating YY1-dependent inhibition. PMID:12023898

Specificity of marine microbial surface interactions.

PubMed Central

Imam, S H; Bard, R F; Tosteson, T R

1984-01-01

The macromolecular surface components involved in intraspecific cell surface interactions of the green microalga Chlorella vulgaris and closely associated bacteria were investigated. The specific surface attachment between this alga and its associated bacteria is mediated by lectin-like macromolecules associated with the surfaces of these cells. The binding activity of these surface polymers was inhibited by specific simple sugars; this suggests the involvement of specific receptor-ligand binding sites on the interactive surfaces. Epifluorescent microscopic evaluation of bacteria-alga interactions in the presence and absence of the macromolecules that mediate these interactions showed that the glycoproteins active in these processes were specific to the microbial sources from which they were obtained. The demonstration and definition of the specificity of these interactions in mixed microbial populations may play an important role in our understanding of the dynamics of marine microbial populations in the sea. PMID:6508293

Concerted Multi-Pronged Attack by Calpastatin to Occlude the Catalytic Cleft of Heterodimeric Calpains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moldoveanu, T.; Gehring, K; Green, D

2008-01-01

The Ca{sup 2+}-dependent cysteine proteases, calpains, regulate cell migration, cell death, insulin secretion, synaptic function and muscle homeostasis. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency. Despite the physiological importance of this interaction, the structural basis of calpain inhibition by calpastatin is unknown. Here we report the 3.0{angstrom} structure of Ca{sup 2+}-bound m-calpain in complex with the first calpastatin repeat, both from rat, revealing the mechanism of exclusive specificity. The structure highlights the complexity of calpain activation by Ca{sup 2+}, illustrating key residues in a peripheral domainmore » that serve to stabilize the protease core on Ca{sup 2+} binding. Fully activated calpain binds ten Ca{sup 2+} atoms, resulting in several conformational changes allowing recognition by calpastatin. Calpain inhibition is mediated by the intimate contact with three critical regions of calpastatin. Two regions target the penta-EF-hand domains of calpain and the third occupies the substrate-binding cleft, projecting a loop around the active site thiol to evade proteolysis.« less

Effect of Escherichia coli DNA binding protein on the transcription of single-stranded phage M13 DNA by Escherichia coli RNA polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niyogi, S.K.; Ratrie, H. III; Datta, A.K.

E. coli DNA binding protein strongly inhibits the transcription of single-stranded rather than double-stranded phage M13 DNA by E. coli RNA polymerase. This inhibition cannot be significantly overcome by increasing the concentration of RNA polymerase. Nor does the order of addition of binding protein affect its inhibitory property: inhibition is evident whether binding protein is added before or after the formation of the RNA polymerase--DNA complex. Inhibition is also observed if binding protein is added at various times after initiation of RNA synthesis. Maximal inhibition occurs at a binding protein-to-DNA ratio (w/w) of about 8:1. This corresponds to one bindingmore » protein molecule covering about 30 nucleotides, in good agreement with values obtained by physical measurements.« less

Efficacy of Lysine-Specific Demethylase 1 Inhibition in PCa

DTIC Science & Technology

2016-08-01

specific demethylase 1 (LSD1) forms a complex with CoREST and has been well-characterized as an epigenetic regulator that mediates transcriptional...castration-resistant prostate cancer (CRPC), where AR activity persists and its function may be altered by epigenetic mechanisms. Specifically, we...hypothesized that LSD1 activity in PCa may allow tumor cells to epigenetically reprogram the AR cistrome by closing AR binding sites through which AR

Competitive Enzyme-Linked Immunosorbent Assay Based on a Rhoptry-Associated Protein 1 Epitope Specifically Identifies Babesia bovis-Infected Cattle

PubMed Central

Goff, Will L.; McElwain, Terry F.; Suarez, Carlos E.; Johnson, Wendell C.; Brown, Wendy C.; Norimine, Junzo; Knowles, Donald P.

2003-01-01

The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool. PMID:12522037

Rational and Modular Design of Potent Ligands Targeting the RNA that Causes Myotonic Dystrophy 2

PubMed Central

Lee, Melissa M.; Pushechnikov, Alexei; Disney, Matthew D.

2009-01-01

Most ligands targeting RNA are identified through screening a therapeutic target for binding members of a ligand library. A potential alternative way to construct RNA binders is through rational design using information about the RNA motifs ligands prefer to bind. Herein, we describe such an approach to design modularly assembled ligands targeting the RNA that causes myotonic dystrophy type 2 (DM2), a currently untreatable disease. A previous study identified that 6′-N-5-hexynoate kanamycin A (1) prefers to bind 2×2 nucleotide, pyrimidine-rich RNA internal loops. Multiple copies of such loops were found in the RNA hairpin that causes DM2. The 1 ligand was then modularly displayed on a peptoid scaffold with varied number and spacing to target several internal loops simultaneously. Modularly assembled ligands were tested for binding to a series of RNAs and for inhibiting the formation of the toxic DM2 RNA-muscleblind protein (MBNL-1) interaction. The most potent ligand displays three 1 modules, each separated by four spacing submonomers, and inhibits the formation of the RNA-protein complex with an IC50 of 25 nM. This ligand is higher affinity and more specific for binding DM2 RNA than MBNL-1. It binds the DM2 RNA at least 20-times more tightly than related RNAs and 15-fold more tightly than MBNL-1. A related control peptoid displaying 6′-N-5-hexynoate neamine (2) is >100-fold less potent at inhibiting the RNA-protein interaction and binds to DM2 RNA >125-fold more weakly. Uptake studies into a mouse myoblast cell line also show that the most potent ligand is cell permeable. PMID:19348464

Binding of endostatin to endothelial heparan sulphate shows a differential requirement for specific sulphates.

PubMed

Blackhall, Fiona H; Merry, Catherine L R; Lyon, Malcolm; Jayson, Gordon C; Folkman, Judah; Javaherian, Kashi; Gallagher, John T

2003-10-01

Endostatin is a naturally occurring proteolytic fragment of the C-terminal domain of collagen XVIII. It inhibits angiogenesis by a mechanism that appears to involve binding to HS (heparan sulphate). We have examined the molecular interaction between endostatin and HS from micro- and macrovessel endothelial cells. Two discrete panels of oligosaccharides were prepared from metabolically radiolabelled HS, using digestion with either heparinase I or III, and then examined for their endostatin affinity using a sensitive filter-binding assay. Two types of endostatin-binding regions were identified: one comprising sulphated domains of five or more disaccharides in length, enriched in 6-O-sulphate groups, and the other contained long heparinase I-resistant fragments. In the latter case, evidence from the present study suggests that the binding region encompasses a sulphated domain fragment and a transition zone of intermediate sulphation. The contribution to binding of specific O-sulphate groups was determined using selectively desulphated HS species, namely HS from Hs2st-/- mutant cells, and by comparing the compositions of endostatin-binding and non-binding oligosaccharides. The results indicate that 6-O-sulphates play a dominant role in site selectivity and 2-O-sulphates are not strictly essential.

Binding of endostatin to endothelial heparan sulphate shows a differential requirement for specific sulphates.

PubMed Central

Blackhall, Fiona H; Merry, Catherine L R; Lyon, Malcolm; Jayson, Gordon C; Folkman, Judah; Javaherian, Kashi; Gallagher, John T

2003-01-01

Endostatin is a naturally occurring proteolytic fragment of the C-terminal domain of collagen XVIII. It inhibits angiogenesis by a mechanism that appears to involve binding to HS (heparan sulphate). We have examined the molecular interaction between endostatin and HS from micro- and macrovessel endothelial cells. Two discrete panels of oligosaccharides were prepared from metabolically radiolabelled HS, using digestion with either heparinase I or III, and then examined for their endostatin affinity using a sensitive filter-binding assay. Two types of endostatin-binding regions were identified: one comprising sulphated domains of five or more disaccharides in length, enriched in 6-O-sulphate groups, and the other contained long heparinase I-resistant fragments. In the latter case, evidence from the present study suggests that the binding region encompasses a sulphated domain fragment and a transition zone of intermediate sulphation. The contribution to binding of specific O-sulphate groups was determined using selectively desulphated HS species, namely HS from Hs2st-/- mutant cells, and by comparing the compositions of endostatin-binding and non-binding oligosaccharides. The results indicate that 6-O-sulphates play a dominant role in site selectivity and 2-O-sulphates are not strictly essential. PMID:12812520

Regulator of differentiation 1 (ROD1) binds to the amphipathic C-terminal peptide of thrombospondin-4 and is involved in its mitogenic activity.

PubMed

Sadvakassova, Gulzhakhan; Dobocan, Monica C; Difalco, Marcos R; Congote, Luis F

2009-09-01

The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin. In the absence of this cytokine, C21 stimulated exclusively myeloid colony formation. Therefore, the peptide is not a specific erythroid differentiation factor. In fact, it is mitogenic in non-erythroid cells, such as skin fibroblasts and kidney epithelial cells. In erythroleukemic TF-1 cells, it actually decreased the production of the erythroid differentiation marker glycophorin A. C21-affinity chromatography revealed regulator of differentiation 1 (ROD1) as a major C21-binding protein. ROD1 is the hematopoietic cell paralog of polypyrimidine tract binding proteins (PTBs), RNA splice regulators which regulate differentiation by repressing tissue-specific exons. ROD1 binding to C21 was strongly inhibited by synthetic RNAs in the order poly A > poly U > poly G = poly C and was weakly inhibited by a synthetic phosphorylated peptide mimicking the C-terminal domain of RNA polymerase II. Cellular overexpression or knockdown experiments of ROD1 suggest a role for this protein in the mitogenic activity of C21. Since the nuclear proteins ROD1 and PTBs regulate differentiation at a posttranscriptional level and there is a fast nuclear uptake of C21, we put forward the idea that the peptide is internalized, goes to the nucleus and maintains cells in a proliferative state by supporting ROD1-mediated inhibition of differentiation.

TRIM25 Is Required for the Antiviral Activity of Zinc Finger Antiviral Protein

PubMed Central

Zheng, Xiaojiao; Wang, Xinlu; Tu, Fan; Wang, Qin; Fan, Zusen

2017-01-01

ABSTRACT Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mRNAs and repressing the translation and/or promoting the degradation of target mRNA. In addition, ZAP regulates the expression of certain cellular genes. Here, we report that tripartite motif-containing protein 25 (TRIM25), a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 abolished ZAP's antiviral activity. The E3 ligase activity of TRIM25 is required for this regulation. TRIM25 mediated ZAP ubiquitination, but the ubiquitination of ZAP itself did not seem to be required for its antiviral activity. Downregulation of endogenous ubiquitin or overexpression of the deubiquitinase OTUB1 impaired ZAP's activity. We provide evidence indicating that TRIM25 modulates the target RNA binding activity of ZAP. These results uncover a mechanism by which the antiviral activity of ZAP is regulated. IMPORTANCE ZAP is a host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1, Sindbis virus, and Ebola virus. ZAP binds directly to target mRNA, and it represses the translation and promotes the degradation of target mRNA. While the mechanisms by which ZAP posttranscriptionally inhibits target RNA expression have been extensively studied, how its antiviral activity is regulated is not very clear. Here, we report that TRIM25, a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 remarkably abolished ZAP's activity. TRIM25 is required for ZAP optimal binding to target mRNA. These results help us to better understand how the antiviral activity of ZAP is regulated. PMID:28202764

TRIM25 Is Required for the Antiviral Activity of Zinc Finger Antiviral Protein.

PubMed

Zheng, Xiaojiao; Wang, Xinlu; Tu, Fan; Wang, Qin; Fan, Zusen; Gao, Guangxia

2017-05-01

Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mRNAs and repressing the translation and/or promoting the degradation of target mRNA. In addition, ZAP regulates the expression of certain cellular genes. Here, we report that tripartite motif-containing protein 25 (TRIM25), a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 abolished ZAP's antiviral activity. The E3 ligase activity of TRIM25 is required for this regulation. TRIM25 mediated ZAP ubiquitination, but the ubiquitination of ZAP itself did not seem to be required for its antiviral activity. Downregulation of endogenous ubiquitin or overexpression of the deubiquitinase OTUB1 impaired ZAP's activity. We provide evidence indicating that TRIM25 modulates the target RNA binding activity of ZAP. These results uncover a mechanism by which the antiviral activity of ZAP is regulated. IMPORTANCE ZAP is a host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1, Sindbis virus, and Ebola virus. ZAP binds directly to target mRNA, and it represses the translation and promotes the degradation of target mRNA. While the mechanisms by which ZAP posttranscriptionally inhibits target RNA expression have been extensively studied, how its antiviral activity is regulated is not very clear. Here, we report that TRIM25, a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 remarkably abolished ZAP's activity. TRIM25 is required for ZAP optimal binding to target mRNA. These results help us to better understand how the antiviral activity of ZAP is regulated. Copyright © 2017 American Society for Microbiology.

CD45-mediated signaling pathway is involved in Rhizoctonia bataticola lectin (RBL)-induced proliferation and Th1/Th2 cytokine secretion in human PBMC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pujari, Radha; Eligar, Sachin M.; Kumar, Natesh

2012-03-23

Highlights: Black-Right-Pointing-Pointer RBL, a potent mitogenic and complex N-glycan specific lectin binds to CD45 on PBMC. Black-Right-Pointing-Pointer RBL triggers CD45-mediated signaling involved in activation of p38MAPK and STAT-5. Black-Right-Pointing-Pointer Inhibition of CD45 PTPase signaling blocks RBL-induced ZAP70 phosphorylation. Black-Right-Pointing-Pointer RBL-CD45 mediated signaling is crucial for RBL-induced immunodulatory activities. -- Abstract: We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate themore » involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.« less

Development of novel decoy oligonucleotides: advantages of circular dumb-bell decoy.

PubMed

Tomita, Naruya; Tomita, Tetsuya; Yuyama, Kazuhiko; Tougan, Takahiro; Tajima, Tsuyoshi; Ogihara, Toshio; Morishita, Ryuichi

2003-04-01

The inhibition of specific transcription regulatory proteins is a novel approach to regulate gene expression. The transcriptional activities of DNA binding proteins can be inhibited by the use of double-stranded oligonucleotides (ODNs) that compete for binding to their specific target sequences in promoters and enhancers. Transfection of this cis-element double-stranded ODN, referred to as decoy ODN, has been reported to be a powerful tool that provides a new class of anti-gene strategies to gene therapy and permits examination of specific gene regulation. We have demonstrated the usefulness of this decoy ODN strategy in animal models of restenosis, myocardial infarction, glomerulonephritis and rheumatoid arthritis. However, one of the major limitations of decoy ODN technology is the rapid degradation of phosphodiester ODNs by intracellular nucleases. To date, several different types of double-stranded decoy ODNs have been developed to overcome this issue. Circular dumb-bell (CD) double-stranded decoy ODNs that were developed to resolve this issue have attracted a high level of interest. In this review, the applications of decoy ODN strategy and the advantages of modified CD double-stranded decoy ODNs will be discussed.

The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

PubMed

Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

2014-12-18

Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Selective inhibition by dactinomycin of NANC sensory bronchoconstriction and [125I]NKA binding due to NK-2 receptor antagonism.

PubMed

Lou, Y P; Delay-Goyet, P; Lundberg, J M

1992-03-01

In the present study, dactinomycin (10(-5) M) inhibited the non-adrenergic, non-cholinergic bronchoconstriction upon antidromic vagal nerve stimulation (1 Hz for 1 min) in the isolated perfused guinea-pig lung by 84%. The release of calcitonin gene-related peptide was unchanged, however, suggesting a postjunctional action. Dactinomycin (10(-5), 5 x 10(-5) M) also reduced non-adrenergic non-cholinergic bronchial contractions (maximally by 75%) induced by electrical field stimulation or capsaicin, while the cholinergic component and non-adrenergic non-cholinergic relaxation remained intact. The neurokinin-2 receptor antagonist L-659,877 (10(-6) M) had a similar effect as dactinomycin, inhibiting the non-adrenergic non-cholinergic bronchial contractions by 69%, while the neurokinin-1 receptor antagonist CP-96,345 (10(-6) M) had no effect. The bronchoconstriction evoked by neurokinin A, the selective neurokinin-2 receptor agonist Nle10neurokinin A (4-10) and capsaicin was markedly inhibited by dactinomycin while the contraction induced by substance P (SP), the selective neurokinin-1 receptor agonist Sar9Met(O2)11SP, endothelin-1 and acetylcholine was not affected. In autoradiographic experiments on guinea-pig lung, [125I]neurokinin A-labelled sections showed dense binding in the bronchial smooth muscle layer. Dactinomycin inhibited the specific binding of [125I]neurokinin A in a concentration-dependent manner (IC50 = 6.3 x 10(-6) M) and 66% of [125I]neurokinin A total binding was inhibited by 10(-4) M dactinomycin. In the rat colon, [125I]neurokinin A binding to neurokinin-2 sites on circular smooth muscle was inhibited by dactinomycin with an IC50 value of 7.9 x 10(-6) M. Dactinomycin failed to reduce increased nerve-evoked contractions or those caused by Nle10neurokinin A (4-10) per se in the rat vas deferens, which are considered to be mediated by neurokinin-2 receptor activation. In the rat portal vein, dactinomycin did not influence the contractions caused by the neurokinin-3 selective agonist Pro7neurokinin B. In conclusion, dactinomycin selectively inhibited neurokinin-2 receptor activation in guinea-pig lung and rat colon, but not in rat vas deferens, which may depend on the existence of different neurokinin-2 receptor subtypes. Neurokinin A is most likely the main endogenous excitatory non-adrenergic non-cholinergic transmitter in guinea-pig bronchi.

Small Molecules Targeting the miRNA-Binding Domain of Argonaute 2: From Computer-Aided Molecular Design to RNA Immunoprecipitation.

PubMed

Bellissimo, Teresa; Masciarelli, Silvia; Poser, Elena; Genovese, Ilaria; Del Rio, Alberto; Colotti, Gianni; Fazi, Francesco

2017-01-01

The development of small-molecule-based target therapy design for human disease and cancer is object of growing attention. Recently, specific microRNA (miRNA) mimicking compounds able to bind the miRNA-binding domain of Argonaute 2 protein (AGO2) to inhibit miRNA loading and its functional activity were described. Computer-aided molecular design techniques and RNA immunoprecipitation represent suitable approaches to identify and experimentally determine if a compound is able to impair the loading of miRNAs on AGO2 protein. Here, we describe these two methodologies that we recently used to select a specific compound able to interfere with the AGO2 functional activity and able to improve the retinoic acid-dependent myeloid differentiation of leukemic cells.

AKI after conditional and kidney-specific knockdown of Stanniocalcin-1

USDA-ARS?s Scientific Manuscript database

Stanniocalcin-1 is an intracrine protein; it binds to the cell surface, is internalized to the mitochondria, and diminishes superoxide generation through induction of uncoupling proteins. In vitro, stanniocalcin-1 inhibits macrophages and preserves endothelial barrier function, and transgenic overex...

Coordination of golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner.

PubMed

Diao, Aipo; Frost, Laura; Morohashi, Yuichi; Lowe, Martin

2008-03-14

During membrane traffic, transport carriers are first tethered to the target membrane prior to undergoing fusion. Mechanisms exist to connect tethering with fusion, but in most cases, the details remain poorly understood. GM130 is a member of the golgin family of coiled-coil proteins tat is involved in membrane tethering at the endoplasmic reticulum (ER) to Golgi intermediate compartment and cis-Golgi. Here, we demonstrate that GM130 interacts with syntaxin 5, a t-SNARE also localized to the early secretory pathway. Binding to syntaxin 5 is specific, direct, and mediated by the membrane-proximal region of GM130. Interestingly, interaction with syntaxin 5 is inhibited by the binding of the vesicle docking protein p115 to a distal binding site in GM130. The interaction between GM130 and the small GTPase Rab1 is also inhibited by p115 binding. Our findings suggest a mechanism for coupling membrane tethering and fusion at the ER to Golgi intermediate compartment and cis-Golgi, with GM130 playing a central role in linking these processes. Consistent with this hypothesis, we find that depletion of GM130 by RNA interference slows the rate of ER to Golgi trafficking in vivo. The interactions of GM130 with syntaxin 5 and Rab1 are also regulated by mitotic phosphorylation, which is likely to contribute to the inhibition of ER to Golgi trafficking that occurs when mammalian cells enter mitosis.

Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

PubMed

Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

2016-09-11

Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

Identification of the hot spot residues for pyridine derivative inhibitor CCT251455 and ATP substrate binding on monopolar spindle 1 (MPS1) kinase by molecular dynamic simulation.

PubMed

Chen, Kai; Duan, Wenxiu; Han, Qianqian; Sun, Xuan; Li, Wenqian; Hu, Shuangyun; Wan, Jiajia; Wu, Jiang; Ge, Yushu; Liu, Dan

2018-03-08

Protein kinase monopolar spindle 1 plays an important role in spindle assembly checkpoint at the onset of mitosis. Over expression of MPS1 correlated with a wide range of human tumors makes it an attractive target for finding an effective and specific inhibitor. In this work, we performed molecular dynamics simulations of protein MPS1 itself as well as protein bound systems with the inhibitor and natural substrate based on crystal structures. The reported orally bioavailable 1 h-pyrrolo [3,2-c] pyridine inhibitors of MPS1 maintained stable binding in the catalytic site, while natural substrate ATP could not stay. Comparative study of stability and flexibility of three systems reveals position shifting of β-sheet region within the catalytic site, which indicates inhibition mechanism was through stabilizing the β-sheet region. Binding free energies calculated with MM-GB/PBSA method shows different binding affinity for inhibitor and ATP. Finally, interactions between protein and inhibitor during molecular dynamic simulations were measured and counted. Residue Gly605 and Leu654 were suggested as important hot spots for stable binding of inhibitor by molecular dynamic simulation. Our results reveal an important position shifting within catalytic site for non-inhibited proteins. Together with hot spots found by molecular dynamic simulation, the results provide important information of inhibition mechanism and will be referenced for designing novel inhibitors.

Actinomyces naeslundii Displays Variant fimP and fimA Fimbrial Subunit Genes Corresponding to Different Types of Acidic Proline-Rich Protein and β-Linked Galactosamine Binding Specificity

PubMed Central

Hallberg, K.; Holm, C.; Öhman, U.; Strömberg, N.

1998-01-01

Actinomyces naeslundii genospecies 1 and 2 bind to acidic proline-rich proteins (APRPs) and statherin via type 1 fimbriae and to β-linked galactosamine (GalNAcβ) structures via type 2 fimbriae. In addition, A. naeslundii displays two types of binding specificity for both APRPs-statherin and GalNAcβ, while Actinomyces odontolyticus binds to unknown structures. To study the molecular basis for these binding specificities, DNA fragments spanning the entire or central portions of fimP (type 1) and fimA (type 2) fimbrial subunit genes were amplified by PCR from strains of genospecies 1 and 2 and hybridized with DNA from two independent collections of oral Actinomyces isolates. Isolates of genospecies 1 and 2 and A. odontolyticus, but no other Actinomyces species, were positive for hybridization with fimP and fimA full-length probes irrespective of binding to APRPs and statherin, GalNAcβ, or unknown structures. Isolates of genospecies 1 and 2, with deviating patterns of GalNAcβ1-3Galα-O-ethyl-inhibitable coaggregation with Streptococcus oralis Ss34 and MPB1, were distinguished by a fimA central probe from genospecies 1 and 2, respectively. Furthermore, isolates of genospecies 1 and 2 displaying preferential binding to APRPs over statherin were positive with a fimP central probe, while a genospecies 2 strain with the opposite binding preference was not. The sequences of fimP and fimA central gene segments were highly conserved among isolates with the same, but diversified between those with a variant, binding specificity. In conclusion, A. naeslundii exhibits variant fimP and fimA genes corresponding to diverse APRP and GalNAcβ specificities, respectively, while A. odontolyticus has a genetically related but distinct adhesin binding specificity. PMID:9712794

Histochemical analysis of glycoconjugates in the skin of a catfish (arius tenuispinis, day).

PubMed

Al-Banaw, A; Kenngott, R; Al-Hassan, J M; Mehana, N; Sinowatz, F

2010-02-01

A histochemical study using conventional carbohydrate histochemistry (periodic-acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish (Arius tenuispinis) was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC-labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose-binding lectins LCA and PSA; the galactosamine-binding lectins DBA, SBA and GLS I; the glucosamine-binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose-binding lectin UEA and the sialic acid-specific lectin SNA. In addition, the galactose-binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N-acetylgalactosamine and N-acetylglucosamine residues.

Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

PubMed Central

Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

2013-01-01

Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. PMID:24013563

Design and characterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maraganore, J.M.; Bourdon, P.; Jablonski, J.

1990-07-31

A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of {alpha}-thrombin. These peptides, called hirulogs, consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 {angstrom} in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 ((D-Phe)-Pro-Arg-Pro-(Gly){sub 4}-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu) inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with K{sub i} = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir{sub 53-64}, which binds to themore » thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with K{sub i} = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. Hirulog-1, but not S-Hir{sub 53-64}, was found to inhibit the incorporation of ({sup 14}C)diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure. Comparison of anticoagulant activities of hirulog-1, hirudin, and S-Hir{sub 53-64} showed that the synthetic hirulog-1 is 2-fold more potent than hirudin and 100-fold more active than S-Hir{sub 53-64} in increasing the activated partial thromboplastin time of normal human plasma.« less

Propensity for HBZ-SP1 isoform of HTLV-I to inhibit c-Jun activity correlates with sequestration of c-Jun into nuclear bodies rather than inhibition of its DNA-binding activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clerc, Isabelle, E-mail: isabelle.clerc@univ-montp1.f; CNRS, UM5236, CPBS, F-34965 Montpellier; Universite Montpellier 2, CPBS, F-34095 Montpellier

2009-09-01

HTLV-I bZIP factor (HBZ) contains a C-terminal zipper domain involved in its interaction with c-Jun. This interaction leads to a reduction of c-Jun DNA-binding activity and prevents the protein from activating transcription of AP-1-dependent promoters. However, it remained unclear whether the negative effect of HBZ-SP1 was due to its weak DNA-binding activity or to its capacity to target cellular factors to transcriptionally-inactive nuclear bodies. To answer this question, we produced a mutant in which specific residues present in the modulatory and DNA-binding domain of HBZ-SP1 were substituted for the corresponding c-Fos amino acids to improve the DNA-binding activity of themore » c-Jun/HBZ-SP1 heterodimer. The stability of the mutant, its interaction with c-Jun, DNA-binding activity of the resulting heterodimer, and its effect on the c-Jun activity were tested. In conclusion, we demonstrate that the repression of c-Jun activity in vivo is mainly due to the HBZ-SP1-mediated sequestration of c-Jun to the HBZ-NBs.« less

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ritvos, O.; Ranta, T.; Jalkanen, J.

1988-05-01

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purifiedmore » 34 K IGF-BP specifically bound (125I)iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of (125I) iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of (125I) iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with (125I)iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-(3H)aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells.« less

Special AT-rich sequence binding protein 1 promotes tumor growth and metastasis of esophageal squamous cell carcinoma.

PubMed

Ma, Jun; Wu, Kaiming; Zhao, Zhenxian; Miao, Rong; Xu, Zhe

2017-03-01

Esophageal squamous cell carcinoma is one of the most aggressive malignancies worldwide. Special AT-rich sequence binding protein 1 is a nuclear matrix attachment region binding protein which participates in higher order chromatin organization and tissue-specific gene expression. However, the role of special AT-rich sequence binding protein 1 in esophageal squamous cell carcinoma remains unknown. In this study, western blot and quantitative real-time polymerase chain reaction analysis were performed to identify differentially expressed special AT-rich sequence binding protein 1 in a series of esophageal squamous cell carcinoma tissue samples. The effects of special AT-rich sequence binding protein 1 silencing by two short-hairpin RNAs on cell proliferation, migration, and invasion were assessed by the CCK-8 assay and transwell assays in esophageal squamous cell carcinoma in vitro. Special AT-rich sequence binding protein 1 was significantly upregulated in esophageal squamous cell carcinoma tissue samples and cell lines. Silencing of special AT-rich sequence binding protein 1 inhibited the proliferation of KYSE450 and EC9706 cells which have a relatively high level of special AT-rich sequence binding protein 1, and the ability of migration and invasion of KYSE450 and EC9706 cells was distinctly suppressed. Special AT-rich sequence binding protein 1 could be a potential target for the treatment of esophageal squamous cell carcinoma and inhibition of special AT-rich sequence binding protein 1 may provide a new strategy for the prevention of esophageal squamous cell carcinoma invasion and metastasis.

Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

PubMed

Shemon, Anne N; Heil, Gary L; Granovsky, Alexey E; Clark, Mathew M; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R; Koide, Shohei

2010-05-05

Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

Connective Tissue Growth Factor Domain 4 Amplifies Fibrotic Kidney Disease through Activation of LDL Receptor-Related Protein 6.

PubMed

Johnson, Bryce G; Ren, Shuyu; Karaca, Gamze; Gomez, Ivan G; Fligny, Cécile; Smith, Benjamin; Ergun, Ayla; Locke, George; Gao, Benbo; Hayes, Sebastian; MacDonnell, Scott; Duffield, Jeremy S

2017-06-01

Connective tissue growth factor (CTGF), a matrix-associated protein with four distinct cytokine binding domains, has roles in vasculogenesis, wound healing responses, and fibrogenesis and is upregulated in fibroblasts and myofibroblasts in disease. Here, we investigated the role of CTGF in fibrogenic cells. In mice, tissue-specific inducible overexpression of CTGF by kidney pericytes and fibroblasts had no bearing on nephrogenesis or kidney homeostasis but exacerbated inflammation and fibrosis after ureteral obstruction. These effects required the WNT receptor LDL receptor-related protein 6 (LRP6). Additionally, pericytes isolated from these mice became hypermigratory and hyperproliferative on overexpression of CTGF. CTGF is cleaved in vivo into distinct domains. Treatment with recombinant domain 1, 1+2 (N terminus), or 4 (C terminus) independently activated myofibroblast differentiation and wound healing responses in cultured pericytes, but domain 4 showed the broadest profibrotic activity. Domain 4 exhibited low-affinity binding to LRP6 in in vitro binding assays, and inhibition of LRP6 or critical signaling cascades downstream of LRP6, including JNK and WNT/ β -catenin, inhibited the biologic activity of domain 4. Administration of blocking antibodies specifically against CTGF domain 4 or recombinant Dickkopf-related protein-1, an endogenous inhibitor of LRP6, effectively inhibited inflammation and fibrosis associated with ureteral obstruction in vivo Therefore, domain 4 of CTGF and the WNT signaling pathway are important new targets in fibrosis. Copyright © 2017 by the American Society of Nephrology.

Biophysical Mechanisms of Endotoxin Neutralization by Cationic Amphiphilic Peptides

PubMed Central

Kaconis, Yani; Kowalski, Ina; Howe, Jörg; Brauser, Annemarie; Richter, Walter; Razquin-Olazarán, Iosu; Iñigo-Pestaña, Melania; Garidel, Patrick; Rössle, Manfred; Martinez de Tejada, Guillermo; Gutsmann, Thomas; Brandenburg, Klaus

2011-01-01

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS. PMID:21641310

Detection of airborne Stachybotrys chartarum macrocyclic trichothecene mycotoxins on particulates smaller than conidia.

PubMed

Brasel, T L; Douglas, D R; Wilson, S C; Straus, D C

2005-01-01

Highly respirable particles (diameter, <1 microm) constitute the majority of particulate matter found in indoor air. It is hypothesized that these particles serve as carriers for toxic compounds, specifically the compounds produced by molds in water-damaged buildings. The presence of airborne Stachybotrys chartarum trichothecene mycotoxins on particles smaller than conidia (e.g., fungal fragments) was therefore investigated. Cellulose ceiling tiles with confluent Stachybotrys growth were placed in gas-drying containers through which filtered air was passed. Exiting particulates were collected by using a series of polycarbonate membrane filters with decreasing pore sizes. Scanning electron microscopy was employed to determine the presence of conidia on the filters. A competitive enzyme-linked immunosorbent assay (ELISA) specific for macrocyclic trichothecenes was used to analyze filter extracts. Cross-reactivity to various mycotoxins was examined to confirm the specificity. Statistically significant (P < 0.05) ELISA binding was observed primarily for macrocyclic trichothecenes at concentrations of 50 and 5 ng/ml and 500 pg/ml (58.4 to 83.5% inhibition). Of the remaining toxins tested, only verrucarol and diacetylverrucarol (nonmacrocyclic trichothecenes) demonstrated significant binding (18.2 and 51.7% inhibition, respectively) and then only at high concentrations. The results showed that extracts from conidium-free filters demonstrated statistically significant (P < 0.05) antibody binding that increased with sampling time (38.4 to 71.9% inhibition, representing a range of 0.5 to 4.0 ng/ml). High-performance liquid chromatography analysis suggested the presence of satratoxin H in conidium-free filter extracts. These data show that S. chartarum trichothecene mycotoxins can become airborne in association with intact conidia or smaller particles. These findings may have important implications for indoor air quality assessment.

Mithramycin inhibits epithelial-to-mesenchymal transition and invasion by downregulating SP1 and SNAI1 in salivary adenoid cystic carcinoma.

PubMed

Li, Jiasu; Gao, Hongmei; Meng, Lingxu; Yin, Lin

2017-06-01

Mithramycin exhibits certain anticancer effects in glioma, metastatic cerebral carcinoma, malignant lymphoma, chorionic carcinoma and breast cancer. However, its effects on salivary adenoid cystic carcinoma remain unclear. Here, we report that mithramycin significantly inhibited epithelial-to-mesenchymal transition and invasion in human salivary adenoid cystic carcinoma cell lines. The underlying mechanism for this activity was further demonstrated to involve decreasing the expression of the transcription factors specificity protein 1 and SNAI1. Specificity protein 1 is a pro-tumourigenic transcription factor that is overexpressed in SACC-LM and SACC-83 cells, and its expression is inhibited by mithramycin. Moreover, chromatin immunoprecipitation assays showed that specificity protein 1 induced SNAI1 transcription through direct binding to the SNAI1 promoter. In summary, this study uncovered the mechanism through which mithramycin inhibits epithelial-to-mesenchymal transition and invasion in salivary adenoid cystic carcinoma cell lines, namely, via downregulating specificity protein 1 and SNAI1 expression, which suggests mithramycin may be a promising therapeutic option for salivary adenoid cystic carcinoma.

Immunization with Hypoallergens of shrimp allergen tropomyosin inhibits shrimp tropomyosin specific IgE reactivity.

PubMed

Wai, Christine Y Y; Leung, Nicki Y H; Ho, Marco H K; Gershwin, Laurel J; Shu, Shang An; Leung, Patrick S C; Chu, Ka Hou

2014-01-01

Designer proteins deprived of its IgE-binding reactivity are being sought as a regimen for allergen-specific immunotherapy. Although shrimp tropomyosin (Met e 1) has long been identified as the major shellfish allergen, no immunotherapy is currently available. In this study, we aim at identifying the Met e 1 IgE epitopes for construction of hypoallergens and to determine the IgE inhibitory capacity of the hypoallergens. IgE-binding epitopes were defined by three online computational models, ELISA and dot-blot using sera from shrimp allergy patients. Based on the epitope data, two hypoallergenic derivatives were constructed by site-directed mutagenesis (MEM49) and epitope deletion (MED171). Nine regions on Met e 1 were defined as the major IgE-binding epitopes. Both hypoallergens MEM49 and MED171 showed marked reduction in their in vitro reactivity towards IgE from shrimp allergy patients and Met e 1-sensitized mice, as well as considerable decrease in induction of mast cell degranulation as demonstrated in passive cutaneous anaphylaxis assay. Both hypoallergens were able to induce Met e 1-recognizing IgG antibodies in mice, specifically IgG2a antibodies, that strongly inhibited IgE from shrimp allergy subjects and Met e 1-sensitized mice from binding to Met e 1. These results indicate that the two designer hypoallergenic molecules MEM49 and MED171 exhibit desirable preclinical characteristics, including marked reduction in IgE reactivity and allergenicity, as well as ability to induce blocking IgG antibodies. This approach therefore offers promises for development of immunotherapeutic regimen for shrimp tropomyosin allergy.

Trim25 Is an RNA-Specific Activator of Lin28a/TuT4-Mediated Uridylation

PubMed Central

Choudhury, Nila Roy; Nowak, Jakub S.; Zuo, Juan; Rappsilber, Juri; Spoel, Steven H.; Michlewski, Gracjan

2014-01-01

Summary RNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis, thereby providing an additional level of specificity. PMID:25457611

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

PubMed Central

Kong, Daochun; Coleman, Thomas R.; DePamphilis, Melvin L.

2003-01-01

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe. PMID:12840006

Digested wheat gluten inhibits binding between leptin and its receptor.

PubMed

Jönsson, Tommy; Memon, Ashfaque A; Sundquist, Kristina; Sundquist, Jan; Olsson, Stefan; Nalla, Amarnadh; Bauer, Mikael; Linse, Sara

2015-01-20

Leptin resistance is considered a primary risk factor for obesity. It has been hypothesized that dietary cereal grain protein could cause leptin resistance by preventing leptin from binding to its receptor. Non-degraded dietary wheat protein has been found in human serum at a mean level of 41 ng/mL. Here, we report our findings from testing whether enzymatically digested gluten from wheat prevents leptin from binding to the leptin receptor in vitro. Gluten from wheat was digested with pepsin and trypsin under physiological conditions. Pepsin and trypsin activity was removed from the gluten digest with a 10 kDa spin-filter or by heat treatment at 100°C for 30 min. Binding to the leptin receptor of leptin mixed with gluten digest at a series of concentrations was measured using surface plasmon resonance technology. Binding of the gluten digest to the leptin receptor was not detected. Spin-filtered gluten digest inhibited binding of leptin to the leptin receptor, with 50% inhibition at a gluten digest concentration of ~10 ng/mL. Heat-treated gluten digest did not inhibit leptin binding. Digested wheat gluten inhibits binding of leptin to the leptin receptor, with half-maximal inhibition at 10 ng/mL. The inhibition is significant at clinically relevant concentrations and could therefore serve as a novel pathway to investigate to understand the molecular basis of leptin resistance, obesity and associated disorders.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Kyungsu; Lee, Kyung-Mi; Yoo, Ji-Hye

Graphical abstract: Schematic diagram of the possible molecular mechanism underlying the inhibition of the Wnt/{beta}-catenin signaling pathway and the induction of G0/G1-phase arrest by gomisins J and N, derived from the fruits of S. chinensis, in HCT116 human colon cancer cells. Highlights: Black-Right-Pointing-Pointer Gomisins J and N inhibited Wnt/{beta}-catenin signaling pathway in HCT116 cells. Black-Right-Pointing-Pointer Gomisins J and N disrupted the binding of {beta}-catenin to specific DNA sequences, TBE. Black-Right-Pointing-Pointer Gomisins J and N inhibited the HCT116 cell proliferation through G0/G1 phase arrest. Black-Right-Pointing-Pointer Gomisins J and N inhibited the expression of Cyc D1, a Wnt/{beta}-catenin target gene. -- Abstract:more » Here, we report that gomisin J and gomisin N, dibenzocyclooctadiene type lignans isolated from Schisandra chinensis, inhibit Wnt/{beta}-catenin signaling in HCT116 cells. Gomisins J and N appear to inhibit Wnt/{beta}-catenin signaling by disrupting the interaction between {beta}-catenin and its specific target DNA sequences (TCF binding elements, TBE) rather than by altering the expression of the {beta}-catenin protein. Gomisins J and N inhibit HCT116 cell proliferation by arresting the cell cycle at the G0/G1 phase. The G0/G1 phase arrest induced by gomisins J and N appears to be caused by a decrease in the expression of Cyclin D1, a representative target gene of the Wnt/{beta}-catenin signaling pathway, as well as Cdk2, Cdk4, and E2F-1. Therefore, gomisins J and N, the novel Wnt/{beta}-catenin inhibitors discovered in this study, may serve as potential agents for the prevention and treatment of human colorectal cancers.« less

Lipid A binding sites in membranes of macrophage tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.

1988-10-15

Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA, which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4'-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay ofmore » immobilized cellular proteins. Using the whole cell assay, we show that 4'-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4'-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.« less

Characterization of digitalis-like factors in human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiac glycoside-specific antibodies.

PubMed

Kelly, R A; O'Hara, D S; Canessa, M L; Mitch, W E; Smith, T W

1985-09-25

Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin-specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma.

Geraniin extracted from the rind of Nephelium lappaceum binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication.

PubMed

Abdul Ahmad, Siti Aisyah; Palanisamy, Uma D; Tejo, Bimo A; Chew, Miaw Fang; Tham, Hong Wai; Syed Hassan, Sharifah

2017-11-21

The rapid rise and spread in dengue cases, together with the unavailability of safe vaccines and effective antiviral drugs, warrant the need to discover and develop novel anti-dengue treatments. In this study the antiviral activity of geraniin, extracted from the rind of Nephelium lappaceum, against dengue virus type-2 (DENV-2) was investigated. Geraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC 50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays'. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay. Cytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC 50 of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin's interaction with rE-DIII with high affinity. Geraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.

Equol is more active than soy isoflavone itself to compete for binding to thromboxane A(2) receptor in human platelets.

PubMed

Muñoz, Yenny; Garrido, Argelia; Valladares, Luis

2009-03-01

Several dietary intervention studies examining the health effect of soy isoflavones allude to the importance of equol in establishing the cardiovascular response to soy protein. Although, the specific mechanism by which this action occurs has not been established. The aim of this study was to investigate the inhibitory effect of soy-isoflavones and the metabolite of daidzein, equol, on agonist-induced platelet responses dependent on thromboxane A(2) (TxA(2)) receptor. Competitive radioligand binding assay was used to screen for affinity of these compounds to the TxA(2) receptor. The effect of equol on platelet activation, evaluate through of release of the ATP, by analogs of TxA(2) was analyzed. The effect of equol on platelet aggregation was investigated with ADP, U46619 (a TxA(2) mimic) and the calcium ionophore A23187. The data showed that aglycone isoflavones and equol bind to TxA(2) receptor in the micromol/L range, whereas their glucoside derivates had very low binding activity for this receptor. Under equilibrium conditions, the following order of the relative affinity in inhibiting [(3)H]-SQ29585 binding was: equol>genistein>daidzein>glycitein>genistin, daidzin, glycitin. Equol interaction was reversible and competitive for labeled-SQ29548 with not apparent decrease in the number of TxA(2) binding sites. In addition, from platelet activation studies, equol effectively inhibited ATP secretion elicited by the TxA(2) analog U46619. On the other hand, equol inhibited the platelet aggregation induced by U46619 and A23187, while it failed to inhibit that induced by ADP. The aglycone isoflavones from soy, and particularly equol, have been found to have biological effects attributable to thromboxane A(2) receptor antagonism. These findings may help elucidate how dietary isoflavone modulate platelet function and explain why soy-rich foods are claimed to have beneficial effects in the prevention of thrombotic events.

Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model

PubMed Central

Takashima, K; Matsunaga, N; Yoshimatsu, M; Hazeki, K; Kaisho, T; Uekata, M; Hazeki, O; Akira, S; Iizawa, Y; Ii, M

2009-01-01

Background and purpose: TAK-242, a novel synthetic small-molecule, suppresses production of multiple cytokines by inhibiting Toll-like receptor (TLR) 4 signalling. In this study, we investigated the target molecule of TAK-242 and examined its therapeutic effect in a mouse sepsis model. Experimental approach: Binding assay with [3H]-TAK-242 and nuclear factor-κB reporter assay were used to identify the target molecule and binding site of TAK-242. Bacillus calmette guerin (BCG)-primed mouse sepsis model using live Escherichia coli was used to estimate the efficacy of TAK-242 in sepsis. Key results: TAK-242 strongly bound to TLR4, but binding to TLR2, 3, 5, 9, TLR-related adaptor molecules and MD-2 was either not observed or marginal. Mutational analysis using TLR4 mutants indicated that TAK-242 inhibits TLR4 signalling by binding to Cys747 in the intracellular domain of TLR4. TAK-242 inhibited MyD88-independent pathway as well as MyD88-dependent pathway and its inhibitory effect was largely unaffected by lipopolysaccharide (LPS) concentration and types of TLR4 ligands. TAK-242 had no effect on the LPS-induced conformational change of TLR4-MD-2 and TLR4 homodimerization. In mouse sepsis model, although TAK-242 alone did not affect bacterial counts in blood, if co-administered with ceftazidime it inhibited the increases in serum cytokine levels and improved survival of mice. Conclusions and implications: TAK-242 suppressed TLR4 signalling by binding directly to a specific amino acid Cys747 in the intracellular domain of TLR4. When co-administered with antibiotics, TAK-242 showed potent therapeutic effects in an E. coli-induced sepsis model using BCG-primed mice. Thus, TAK-242 may be a promising therapeutic agent for sepsis. PMID:19563534

Production of Recombinant Human scFv Against Tetanus Toxin Heavy Chain by Phage Display Technology.

PubMed

Khalili, Ehsan; Lakzaei, Mostafa; Rasaee, Mohhamad Javad; Aminian, Mahdi

2015-10-01

Tetanus, as a major cause of death in developing countries, is caused by tetanus neurotoxin. Recombinant antibodies against tetanus neurotoxin can be useful in tetanus management. Phage display of antibody fragments from immune human antibody libraries with single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. The aim of this study was the generation of a single chain fragment of variable region (scFv) library and selection of specific antibodies with high affinity against tetanus toxin. Immune human single chain fragment variable (HuscFv) antibody phagemid library was displayed on pIII of filamentous bacteriophage. Selection of scFv clones was performed against tetanus toxin antigens after three rounds of panning. The selected scFv clones were analyzed for inhibition of tetanus toxin binding to ganglioside GT1b. After the third round of panning, over 35 HuscFv phages specific for tetanus toxin were isolated from this library of which 15 clones were found to bind specifically to tetanus toxin. The selected HuscFv phages expressed as a soluble HuscFv peptide and some clones showed positive signals against tetanus toxin. We found that six HuscFv clones inhibit toxin binding to ganglioside GT1b. These selected antibodies can be used in the management of tetanus.

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase.

PubMed

Gadda, Giovanni; Powell, Nichole L N; Menon, Prashanthi

2004-10-15

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.

Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum.

PubMed Central

Castagliuolo, I; LaMont, J T; Nikulasson, S T; Pothoulakis, C

1996-01-01

Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans. PMID:8945570

Solubilization and purification of melatonin receptors from lizard brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivkees, S.A.; Conron, R.W. Jr.; Reppert, S.M.

Melatonin receptors in lizard brain were identified and characterized using {sup 125}I-labeled melatonin (({sup 125}I)MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resultedmore » in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.« less

Inhibition of m3 muscarinic acetylcholine receptors by local anaesthetics

PubMed Central

Hollmann, Markus W; Ritter, Carsten H; Henle, Philipp; de Klaver, Manuela; Kamatchi, Ganesan L; Durieux, Marcel E

2001-01-01

Muscarinic m1 receptors are inhibited by local anaesthetics (LA) at nM concentrations. To elucidate in more detail the site(s) of LA interaction, we compared these findings with LA effects on m3 muscarinic receptors. We expressed receptors in Xenopus oocytes. Using two-electrode voltage clamp, we measured the effects of lidocaine, QX314 (permanently charged) and benzocaine (permanently uncharged) on Ca2+-activated Cl−-currents (ICl(Ca)), elicited by acetyl-β-methylcholine bromide (MCh). We also characterized the interaction of lidocaine with [3H]-quinuclydinyl benzylate ([3H]-QNB) binding to m3 receptors. Antisense-injection was used to determine the role of specific G-protein α subunits in mediating the inhibitory effects of LA. Using chimeric receptor constructs we investigated which domains of the muscarinic receptors contribute to the binding site for LA. Lidocaine inhibited m3-signalling in a concentration-dependent, reversible, non-competitive manner with an IC50 of 370 nM, approximately 21 fold higher than the IC50 (18 nM) reported for m1 receptors. Intracellular inhibition of both signalling pathways by LA was similar, and dependent on the Gq- protein α subunit. In contrast to results reported for the m1 receptor, the m3 receptor lacks the major extracellular binding site for charged LA. The N-terminus and third extracellular loop of the m1 muscarinic receptor molecule were identified as requirements to obtain extracellular inhibition by charged LA. PMID:11325812

Syk Inhibits the Activity of Protein Kinase A by Phosphorylating Tyrosine 330 of the Catalytic Subunit*

PubMed Central

Yu, Shuai; Huang, He; Iliuk, Anton; Wang, Wen-Horng; Jayasundera, Keerthi B.; Tao, W. Andy; Post, Carol B.; Geahlen, Robert L.

2013-01-01

The Syk protein-tyrosine kinase can have multiple effects on cancer cells, acting in some as a tumor suppressor by inhibiting motility and in others as a tumor promoter by enhancing survival. Phosphoproteomic analyses identified PKA as a Syk-specific substrate. Syk catalyzes the phosphorylation of the catalytic subunit of PKA (PKAc) both in vitro and in cells on Tyr-330. Tyr-330 lies within the adenosine-binding motif in the C-terminal tail of PKAc within a cluster of acidic amino acids (DDYEEEE), which is a characteristic of Syk substrates. The phosphorylation of PKAc on Tyr-330 by Syk strongly inhibits its catalytic activity. Molecular dynamics simulations suggest that this additional negative charge prevents the C-terminal tail from interacting with the substrate and the nucleotide-binding site to stabilize the closed conformation of PKAc, thus preventing catalysis from occurring. Phosphoproteomic analyses and Western blotting studies indicate that Tyr-330 can be phosphorylated in a Syk-dependent manner in MCF7 breast cancer cells and DT40 B cells. The phosphorylation of a downstream substrate of PKAc, cAMP-responsive element-binding protein (CREB), is inhibited in cells expressing Syk but can be rescued by a selective inhibitor of Syk. Modulation of CREB activity alters the expression of the CREB-regulated gene BCL2 and modulates cellular responses to genotoxic agents. Thus, PKA is a novel substrate of Syk, and its phosphorylation on Tyr-330 inhibits its participation in downstream signaling pathways. PMID:23447535

Ketoconazole inhibition of testicular secretion of testosterone and displacement of steroid hormones from serum transport proteins.

PubMed Central

Grosso, D S; Boyden, T W; Pamenter, R W; Johnson, D G; Stevens, D A; Galgiani, J N

1983-01-01

In vivo perfusion of canine testes with ketoconazole inhibited the stimulation of testosterone production by human chorionic gonadotropin in a dose-dependent manner. Ketoconazole also selectively displaced steroids from serum-binding globulins. Dihydrotestosterone and estradiol binding to sex hormone-binding globulin were inhibited by ketoconazole. Cortisol binding to corticosteroid-binding globulin was unaffected. The concentrations of ketoconazole that inhibited human chorionic gonadotropin stimulation of testicular androgen production and displaced sex steroids from sex hormone-binding globulin were in the range of blood levels found in patients on higher therapeutic dosage regimens. Suppression of testicular testosterone synthesis and displacement of estrogens from sex hormone-binding globulin may decrease the androgen/estrogen ratio of the blood and contribute to the development of gynecomastia that has been reported in some ketoconazole-treated patients. PMID:6301363

Molecular Dynamics Simulation Study on the Binding and Stabilization Mechanism of Antiprion Compounds to the "Hot Spot" Region of PrPC.

PubMed

Zhou, Shuangyan; Liu, Xuewei; An, Xiaoli; Yao, Xiaojun; Liu, Huanxiang

2017-11-15

Structural transitions in the prion protein from the cellular form, PrP C , into the pathological isoform, PrP Sc , are regarded as the main cause of the transmissible spongiform encephalopathies, also known as prion diseases. Hence, discovering and designing effective antiprion drugs that can inhibit PrP C to PrP Sc conversion is regarded as a promising way to cure prion disease. Among several strategies to inhibit PrP C to PrP Sc conversion, stabilizing the native PrP C via specific binding is believed to be one of the valuable approaches and many antiprion compounds have been reported based on this strategy. However, the detailed mechanism to stabilize the native PrP C is still unknown. As such, to unravel the stabilizing mechanism of these compounds to PrP C is valuable for the further design and discovery of antiprion compounds. In this study, by molecular dynamics simulation method, we investigated the stabilizing mechanism of several antiprion compounds on PrP C that were previously reported to have specific binding to the "hot spot" region of PrP C . Our simulation results reveal that the stabilization mechanism of specific binding compounds can be summarized as (I) to stabilize both the flexible C-terminal of α2 and the hydrophobic core, such as BMD42-29 and GN8; (II) to stabilize the hydrophobic core, such as J1 and GJP49; (III) to stabilize the overall structure of PrP C by high binding affinity, as NPR-056. In addition, as indicated by the H-bond analysis and decomposition analysis of binding free energy, the residues N159 and Q160 play an important role in the specific binding of the studied compounds and all these compounds interact with PrP C in a similar way with the key interacting residues L130 in the β1 strand, P158, N159, Q160, etc. in the α1-β2 loop, and H187, T190, T191, etc. in the α2 C-terminus although the compounds have large structural difference. As a whole, our obtained results can provide some insights into the specific binding mechanism of main antiprion compounds to the "hot spot" region of PrP C at the molecular level and also provide guidance for effective antiprion drug design in the future.

Bestatin Inhibits Cell Growth, Cell Division, and Spore Cell Differentiation in Dictyostelium discoideum

PubMed Central

Poloz, Yekaterina; Catalano, Andrew

2012-01-01

Bestatin methyl ester (BME) is an inhibitor of Zn2+-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have used Dictyostelium as a model organism to study the effects of BME. Only two Zn2+-binding aminopeptidases have been identified in Dictyostelium to date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development of Dictyostelium, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation in Dictyostelium likely through inhibition of PsaA. PMID:22345351

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP)

PubMed Central

Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R. Max; Tu, Benjamin P.; MacMillan, John B.; De Brabander, Jef K.; Veech, Richard L.; Uyeda, Kosaku

2016-01-01

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. PMID:26984404

A novel multiplex poliovirus binding inhibition assay applicable for large serosurveillance and vaccine studies, without the use of live poliovirus.

PubMed

Schepp, Rutger M; Berbers, Guy A M; Ferreira, José A; Reimerink, Johan H; van der Klis, Fiona R

2017-03-01

Large-scale serosurveillance or vaccine studies for poliovirus using the "gold standard" WHO neutralisation test (NT) are very laborious and time consuming. With the polio eradication at hand and with the removal of live attenuated Sabin strains from the oral poliovirus vaccine (OPV), starting with type 2 (as of April 2016), laboratories will need to conform to much more stringent laboratory biosafety regulations when handling live poliovirus strains. In this study, a poliovirus binding inhibition multiplex immunoassay (polio MIA) using inactivated poliovirus vaccine (IPV-Salk) was developed for simultaneous quantification of serum antibodies directed to all three poliovirus types. Our assay shows a good correlation with the NT and an excellent correlation with the ELISA-based binding inhibition assay (POBI). The assay is highly type-specific and reproducible. Additionally, serum sample throughput increases about fivefold relative to NT and POBI and the amount of serum needed is reduced by more than 90%. In conclusion, the polio MIA can be used as a safe and high throughput application, especially for large-scale surveillance and vaccine studies, reducing laboratory time and serum amounts needed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Elevated HLA-A expression impairs HIV control through inhibition of NKG2A-expressing cells.

PubMed

Ramsuran, Veron; Naranbhai, Vivek; Horowitz, Amir; Qi, Ying; Martin, Maureen P; Yuki, Yuko; Gao, Xiaojiang; Walker-Sperling, Victoria; Del Prete, Gregory Q; Schneider, Douglas K; Lifson, Jeffrey D; Fellay, Jacques; Deeks, Steven G; Martin, Jeffrey N; Goedert, James J; Wolinsky, Steven M; Michael, Nelson L; Kirk, Gregory D; Buchbinder, Susan; Haas, David; Ndung'u, Thumbi; Goulder, Philip; Parham, Peter; Walker, Bruce D; Carlson, Jonathan M; Carrington, Mary

2018-01-05

The highly polymorphic human leukocyte antigen ( HLA ) locus encodes cell surface proteins that are critical for immunity. HLA-A expression levels vary in an allele-dependent manner, diversifying allele-specific effects beyond peptide-binding preference. Analysis of 9763 HIV-infected individuals from 21 cohorts shows that higher HLA-A levels confer poorer control of HIV. Elevated HLA-A expression provides enhanced levels of an HLA-A-derived signal peptide that specifically binds and determines expression levels of HLA-E, the ligand for the inhibitory NKG2A natural killer (NK) cell receptor. HLA-B haplotypes that favor NKG2A-mediated NK cell licensing (i.e., education) exacerbate the deleterious effect of high HLA-A on HIV control, consistent with NKG2A-mediated inhibition impairing NK cell clearance of HIV-infected targets. Therapeutic blockade of HLA-E:NKG2A interaction may yield benefit in HIV disease. Copyright © 2017, American Association for the Advancement of Science.

Saccharin and Cyclamate Inhibit Binding of Epidermal Growth Factor

NASA Astrophysics Data System (ADS)

Lee, L. S.

1981-02-01

The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.

Influence of HLA-DR and -DQ alleles on autoantibody recognition of distinct epitopes within the juxtamembrane domain of the IA-2 autoantigen in type 1 diabetes.

PubMed

Richardson, Carolyn C; McLaughlin, Kerry A; Morgan, Diana; Feltbower, Richard G; Christie, Michael R

2016-02-01

Insulinoma-associated protein 2 (IA-2) is a major target of autoimmunity in type 1 diabetes. When first detected, IA-2-autoantibodies commonly bind epitopes in the juxtamembrane (JM) domain of IA-2 and antibody responses subsequently spread to the tyrosine phosphatase domain. Definition of structures of epitopes in the JM domain, and genetic requirements for autoimmunity to these epitopes, is important for our understanding of initiation and progression of autoimmunity. The aims of this study were to investigate the contribution of individual amino acids in the IA-2 JM domain to antibody binding to these epitopes and the role of HLA genotypes in determining epitope specificity. Regions of the JM domain recognised by autoantibodies were identified by peptide competition and inhibitory effects of alanine substitutions of residues within the JM region. Antibody binding was determined by radioligand binding assays using sera from patients genotyped for HLA-DRB1 and -DQB1 alleles. Patients were categorised into two distinct groups of JM antibody reactivity according to peptide inhibition. Inhibition by substitutions of individual amino acids within the JM domain differed between patients, indicating heterogeneity in epitope recognition. Cluster analysis defined six groups of residues having similar inhibitory effects on antibody binding, with three clusters showing differences in patients affected or unaffected by peptide. One cluster demonstrated significant differences in antibody binding between HLA-DRB1*04 and HLA-DRB1*07 patients and within DRB1*04 individuals; antibody recognition of a second cluster depended on expression of HLA-DQB1*0302. The results identify amino acids contributing to distinct epitopes on IA-2, with both HLA-DR and HLA-DQ alleles influencing epitope specificity.

Modeling the Binding of Neurotransmitter Transporter Inhibitors with Molecular Dynamics and Free Energy Calculations

NASA Astrophysics Data System (ADS)

Jean, Bernandie

The monoamine transporter (MAT) proteins responsible for the reuptake of the neurotransmitter substrates, dopamine, serotonin, and norepinephrine, are drug targets for the treatment of psychiatric disorders including depression, anxiety, and attention deficit hyperactivity disorder. Small molecules that inhibit these proteins can serve as useful therapeutic agents. However, some dopamine transporter (DAT) inhibitors, such as cocaine and methamphetamine, are highly addictive and abusable. Efforts have been made to develop small molecules that will inhibit the transporters and elucidate specific binding site interactions. This work provides knowledge of molecular interactions associated with MAT inhibitors by offering an atomistic perspective that can guide designs of new pharmacotherapeutics with enhanced activity. The work described herein evaluates intermolecular interactions using computational methods to reveal the mechanistic detail of inhibitors binding in the DAT. Because cocaine recognizes the extracellular-facing or outward-facing (OF) DAT conformation and benztropine recognizes the intracellular-facing or inward-facing (IF) conformation, it was postulated that behaviorally "typical" (abusable, locomotor psychostimulant) inhibitors stabilize the OF DAT and "atypical" (little or no abuse potential) inhibitors favor IF DAT. Indeed, behaviorally-atypical cocaine analogs have now been shown to prefer the OF DAT conformation. Specifically, the binding interactions of two cocaine analogs, LX10 and LX11, were studied in the OF DAT using molecular dynamics simulations. LX11 was able to interact with residues of transmembrane helix 8 and bind in a fashion that allowed for hydration of the primary binding site (S1) from the intracellular space, thus impacting the intracellular interaction network capable of regulating conformational transitions in DAT. Additionally, a novel serotonin transporter (SERT) inhibitor previously discovered through virtual screening at the SERT secondary binding site (S2) was studied. Intermolecular interactions between SM11 and SERT have been assessed using binding free energy calculations to predict the ligand-binding site and optimize ligand-binding interactions. Results indicate the addition of atoms to the 4-chlorobenzyl moiety were most energetically favorable. The simulations carried out in DAT and SERT were supported by experimental results. Furthermore, the co-crystal structures of DAT and SERT share similar ligand-binding interactions with the homology models used in this study.

Muscarinic Receptor Binding in Rat Bladder Urothelium and Detrusor Muscle by Intravesical Solifenacin.

PubMed

Ito, Yoshihiko; Kashiwabara, Michishi; Yoshida, Akira; Hikiyama, Eriko; Onoue, Satomi; Yamada, Shizuo

2016-01-01

Solifenacin is an antimuscarinic agent used to treat symptoms of overactive bladder. Pharmacologically significant amounts of solifenacin were excreted in the urine of humans taking a clinical dose of this drug. The aim of this study is to measure muscarinic receptor binding in the bladder urothelium and detrusor muscles of rats following the intravesical instillation of solifenacin. Muscarinic receptors were measured by radioreceptor assay using [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS), a selective radioligand of muscarinic receptors. Solifenacin showed concentration-dependent inhibition of specific [(3)H]NMS binding in the bladder urothelium and detrusor muscle of rats, with no significant difference in Ki values or Hill coefficients between these tissues. Following the intravesical instillation of solifenacin, there was significant muscarinic receptor binding (increase in Kd for specific [(3)H]NMS binding) in the bladder urothelium and detrusor muscle of rats. Similar bladder muscarinic receptor binding was observed by the intravesical instillation of oxybutynin, but not with trospium. In conclusion, the present study has demonstrated that solifenacin binds muscarinic receptors not only in the detrusor muscle but also in the bladder urothelium with high affinity. These bladder muscarinic receptors may be significantly affected by solifenacin excreted in the urine.

Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bencherif, M.; Lukas, R.J.

1991-06-01

Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10more » microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.« less

A novel compound inhibits rHDL assembly and blocks nascent HDL biogenesis downstream of apoAI binding to ABCA1 expressing cells

PubMed Central

Lyssenko, Nicholas N.; Brubaker, Gregory; Smith, Bradley D.; Smith, Jonathan D.

2011-01-01

Objective Nascent high-density lipoprotein (HDL) particles form from cellular lipids and extracellular lipid-free apolipoprotein AI (apoAI) in a process mediated by ATP-binding cassette transporter A1 (ABCA1). We have sought out compounds that inhibit nascent HDL biogenesis without affecting ABCA1 activity. Methods and Results Reconstituted HDL (rHDL) formation and cellular cholesterol efflux assays were used to show that two compounds that bond via hydrogen with phospholipids inhibit rHDL and nascent HDL production. In rHDL formation assays, the inhibitory effect of compound 1 (methyl 3α-acetoxy-7α,12α-di[(phenylaminocarbonyl)amino]-5β-cholan-24-oate), the more active of the two, depended on its ability to associate with phospholipids. In cell assays, compound 1 suppressed ABCA1-mediated cholesterol efflux to apoAI, the 18A peptide, and taurocholate with high specificity, without affecting ABCA1-independent cellular cholesterol efflux to HDL and endocytosis of acetylated low-density lipoprotein (AcLDL) and transferrin. Furthermore, compound 1 did not affect ABCA1 activity adversely, as ABCA1-mediated shedding of microparticles proceeded unabated and apoAI binding to ABCA1-expressing cells increased in its presence. Conclusions The inhibitory effects of compound 1 support a three-step model of nascent HDL biogenesis: plasma membrane remodeling by ABCA1, apoAI binding to ABCA1, and lipoprotein particle assembly. The compound inhibits the final step, causing accumulation of apoAI in ABCA1-expressing cells. PMID:21836073

Monoclonal Antibodies to the V2 Domain of MN-rgp120: Fine Mapping of Epitopes and Inhibition of α4β7 Binding

PubMed Central

Nakamura, Gerald R.; Fonseca, Dora P. A. J.; O'Rourke, Sara M.; Vollrath, Aaron L.; Berman, Phillip W.

2012-01-01

Background Recombinant gp120 (MN-rgp120) was a major component of the AIDSVAX B/E vaccine used in the RV144 trial. This was the first clinical trial to show that vaccination could prevent HIV infection in humans. A recent RV144 correlates of protection study found that protection correlated with the presence of antibodies to the V2 domain. It has been proposed that antibodies to the α4β7 binding site in the V2 domain might prevent HIV-1 infection by blocking the ability of virions to recognize α4β7 on activated T-cells. In this study we investigated the specificity of monoclonal antibodies (MAbs) to the V2 domain of MN-rgp120 and examined the possibility that these antibodies could inhibit the binding of MN-rgp120 to the α4β7 integrin. Methodology/Principal Findings Nine MAbs to the V2 domain were isolated from mice immunized with recombinant envelope proteins. The ability of these MAbs to inhibit HIV infection, block the binding of gp120 to CD4, and block the binding of MN-rgp120 to the α4β7 integrin was measured. Mutational analysis showed that eight of the MAbs recognized two immunodominant clusters of amino acids (166–168 and 178–183) located at either end of the C strand within the four-strand anti-parallel sheet structure comprising the V1/V2 domain. Conclusions/Significance These studies showed that the antigenic structure of the V2 domain is exceedingly complex and that MAbs isolated from mice immunized with MN-rgp120 exhibited a high level of strain specificity compared to MAbs to the V2 domain isolated from HIV-infected humans. We found that immunization with MN-rgp120 readily elicits antibodies to the V2 domain and some of these were able to block the binding of MN-rgp120 to the α4β7 integrin. PMID:22720026

Production of Antiserum to LH-Releasing Hormone (LH-RH) Associated with Gonadal Atrophy in Rabbits: Development of Radioimmunoassays for LH-RH

DOE Office of Scientific and Technical Information (OSTI.GOV)

ARIMURA, A.; SATO, H.; KUMASAKA, T.

1973-11-01

Repeated injections of synthetic LH -- RH decapeptide, adsorbed on polyvinylpyrrolidone and emulsified with complete Freund's adjuvant, resulted in the production of a specific antiserum to LH-- RH in two of three rabbits. The animals that produced this antiserum showed a reduction of pituitary LH content and marked atrophy of the testes. The antiserum-antibody complex was detected by the complement flxation test. The antiserum was capable of binding /sup 125/I- labeled LH--RH. After iodination of LHRH (using /sup 125/I and either the chloramine T or lactoperoxidase method) separation of the iodination products on CMC yielded three main peaks of radioactivity:more » The first was free iodide, the second was labeled peptide with low immunoreactivity, and the third was immunoreactive peptide. This 3rd peak consisted of two or three subpeaks; the leading subpeak(s) were more readily bound by antiserum than the trailing one(s). Binding of these fractions to antiserum was increased in the presence of small amounts of unlabeled LH--RH (a phenomenon called paradoxical binding or hock effect) but inhibited by larger amounts. Both the augmentation and the inhibition effects were dose-related, allowing the development of two different radioimmunoassay (RIA) systems for LH--RH. An ordinary (coinpetitive) type of RIA was developed in which a small amount (0.31 ng/assay tube) of unlabeled LH-- RH was added to the labeled peptide. This saturated the antiserum's capacity for paradoxical binding, so that further addition of LH-- RH (from 0.04 to 2.5 ng/ tube) inhibited binding of labeled LH--RH. The assay developed using paradoxical binding omitted the premixing of labeled and unlabeled LH--RH; in this assay addition of very small amounts (0.5 to 310 pg) of unlabeled LH--RH to the assay tubes increased the amount of label bound to antiserum and allowed construction of a parabolic curve of positive slope when B/T was plotted against arithmetic dose. The assays seem to be highly specific for LH--RH although both polymers and degradation products of LH--RH appeared to have some immunoreactivity.« less

Inhibition of eukaryotic translation elongation by the antitumor natural product Mycalamide B.

PubMed

Dang, Yongjun; Schneider-Poetsch, Tilman; Eyler, Daniel E; Jewett, John C; Bhat, Shridhar; Rawal, Viresh H; Green, Rachel; Liu, Jun O

2011-08-01

Mycalamide B (MycB) is a marine sponge-derived natural product with potent antitumor activity. Although it has been shown to inhibit protein synthesis, the molecular mechanism of action by MycB remains incompletely understood. We verified the inhibition of translation elongation by in vitro HCV IRES dual luciferase assays, ribosome assembly, and in vivo [(35)S]methinione labeling experiments. Similar to cycloheximide (CHX), MycB inhibits translation elongation through blockade of eEF2-mediated translocation without affecting the eEF1A-mediated loading of tRNA onto the ribosome, AUG recognition, or dipeptide synthesis. Using chemical footprinting, we identified the MycB binding site proximal to the C3993 28S rRNA residue on the large ribosomal subunit. However, there are also subtle, but significant differences in the detailed mechanisms of action of MycB and CHX. First, MycB arrests the ribosome on the mRNA one codon ahead of CHX. Second, MycB specifically blocked tRNA binding to the E-site of the large ribosomal subunit. Moreover, they display different polysome profiles in vivo. Together, these observations shed new light on the mechanism of inhibition of translation elongation by MycB.

Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Kyung-Mi; Yun, Ji Ho; Lee, Dong Hwa

2015-04-17

We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cellmore » proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus.« less

The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain*

PubMed Central

Podolnikova, Nataly P.; Yakovlev, Sergiy; Yakubenko, Valentin P.; Wang, Xu; Gorkun, Oleg V.; Ugarova, Tatiana P.

2014-01-01

The currently available antithrombotic agents target the interaction of platelet integrin αIIbβ3 (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of αIIbβ3 antagonists; however, the mechanisms by which αIIbβ3 binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for αIIbβ3 involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the αIIb β-propeller domain of αIIbβ3 enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the αIIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant αIIbβ3 receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin αMβ2 exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the αIIb β-propeller and further indicate that recognition specificity of αIIbβ3 for fibrin differs from that for soluble fibrinogen. PMID:24338009

A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis

PubMed Central

Fukunaga, Ryuya; Zamore, Phillip D

2014-01-01

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme. PMID:24787225

Binding of sulphonated indigo derivatives to RepA-WH1 inhibits DNA-induced protein amyloidogenesis

PubMed Central

Gasset-Rosa, Fátima; Maté, María Jesús; Dávila-Fajardo, Cristina; Bravo, Jerónimo; Giraldo, Rafael

2008-01-01

The quest for inducers and inhibitors of protein amyloidogenesis is of utmost interest, since they are key tools to understand the molecular bases of proteinopathies such as Alzheimer, Parkinson, Huntington and Creutzfeldt–Jakob diseases. It is also expected that such molecules could lead to valid therapeutic agents. In common with the mammalian prion protein (PrP), the N-terminal Winged-Helix (WH1) domain of the pPS10 plasmid replication protein (RepA) assembles in vitro into a variety of amyloid nanostructures upon binding to different specific dsDNA sequences. Here we show that di- (S2) and tetra-sulphonated (S4) derivatives of indigo stain dock at the DNA recognition interface in the RepA-WH1 dimer. They compete binding of RepA to its natural target dsDNA repeats, found at the repA operator and at the origin of replication of the plasmid. Calorimetry points to the existence of a major site, with micromolar affinity, for S4-indigo in RepA-WH1 dimers. As revealed by electron microscopy, in the presence of inducer dsDNA, both S2/S4 stains inhibit the assembly of RepA-WH1 into fibres. These results validate the concept that DNA can promote protein assembly into amyloids and reveal that the binding sites of effector molecules can be targeted to inhibit amyloidogenesis. PMID:18285361

Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions.

PubMed Central

Grober, J S; Bowen, B L; Ebling, H; Athey, B; Thompson, C B; Fox, D A; Stoolman, L M

1993-01-01

Blood monocytes are the principal reservoir for tissue macrophages in rheumatoid synovitis. Receptor-mediated adhesive interactions between circulating cells and the synovial venules initiate recruitment. These interactions have been studied primarily in cultured endothelial cells. Thus the functional activities of specific adhesion receptors, such as the endothelial selectins and the leukocytic integrins, have not been evaluated directly in diseased tissues. We therefore examined monocyte-microvascular interactions in rheumatoid synovitis by modifying the Stamper-Woodruff frozen section binding assay initially developed to study lymphocyte homing. Specific binding of monocytes to venules lined by low or high endothelium occurred at concentrations as low as 5 x 10(5) cells/ml. mAbs specific for P-selectin (CD62, GMP-140/PADGEM) blocked adhesion by > 90% in all synovitis specimens examined. In contrast, P-selectin-mediated adhesion to the microvasculature was either lower or absent in frozen sections of normal foreskin and placenta. mAbs specific for E-selectin (ELAM-1) blocked 20-50% of monocyte attachment in several RA synovial specimens but had no effect in others. mAbs specific for LFA-1, Mo1/Mac 1, the integrin beta 2-chain, and L-selectin individually inhibited 30-40% of adhesion. An mAb specific for the integrin beta 1-chain inhibited the attachment of elutriated monocytes up to 20%. We conclude that P-selectin associated with the synovial microvasculature initiates shear-resistant adhesion of monocytes in the Stamper-Woodruff assay and stabilizes bonds formed by other selectins and the integrins. Thus the frozen section binding assay permits direct evaluation of leukocyte-microvascular adhesive interactions in inflamed tissues and suggests a prominent role for P-selectin in monocyte recruitment in vivo. Images PMID:7685772

The influence of surface integrin binding patterns on specific biomaterial-cell interactions

NASA Astrophysics Data System (ADS)

Beranek, Maggi Marie

As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second aim, the response of surface-bound integrins to flow-related shear stress was examined. Based on fluorescent analysis, total alphavbeta 3, alpha1beta1, and alpha5beta 1 appeared to increase on stainless steel after 90-minute low shear stress exposure, whereas only alpha5beta1 appeared to increase when exposed to high shear. 65/35 poly(D, L-lactide-co-glycolide) exhibited increased total binding of alpha5beta1 and alphaMbeta2, when exposed to either shear stress level. Exposure to either shear stress regimen appeared to increase binding of all integrins on the combinational surface. These responses to shear stress suggest differential integrin binding affinity compared to stainless steel. Using antibodies specific to the integrin subunits, the apparent increase in surface-bound integrins was found to be related to a surface disassociation of alpha and beta subunits. The third aim evaluated human aortic endothelial cells and acute monocytic leukemia cells (THP-1) cell binding to the tested biomaterial surfaces under both static and flow conditions. Both stainless steel and the combinational surface had increased endothelial cell binding compared to monocyte attachment. Pre-incubation of the surface with the specific integrins significantly inhibited human aortic endothelial cell binding. Aim four was designed to investigate the influence of surface bound integrins on human aortic endothelial cell migration under shear stress. If biomaterial surface integrin binding patterns are specific, then pre-bound surface integrins should competitively inhibit binding of cellular integrins to the surface. Cell migration distance on to alphavbeta3, alpha 1beta1, and alpha5beta1 pre-incubated stainless steel was decreased ten-fold, and decreased by three-fold on both 65/35 poly(D, L-lactide-coglycolide) and combinational surfaces compared to the respective bare surfaces. In contrast, migration distance on to alphaMbeta2 pre-coated stainless steel and combinational surface was decreased by only sixty percent and only fifty percent on alphaMbeta2 precoated 65/35 poly(D, L -lactide-co-glycolide). These results suggested that surface binding sites are selective and critical in governing endothelial cell migration. In conclusion, these results support the hypothesis that a surface that encourages specific integrin binding would promote differential cell binding. The novel integrin binding model used in this investigation may be a methodology that can be employed to evaluate potential vascular biomaterials.

A novel substance P binding site in bovine adrenal medulla.

PubMed

Geraghty, D P; Livett, B G; Rogerson, F M; Burcher, E

1990-05-04

Radioligand binding techniques were used to characterize the substance P (SP) binding site on membranes prepared from bovine adrenal medullae. 125I-labelled Bolton-Hunter substance P (BHSP), which recognises the C-terminally directed, SP-preferring NK1 receptor, showed no specific binding. In contrast, binding of [3H]SP was saturable (at 6 nM) and reversible, with an equilibrium dissociation constant (Kd) 1.46 +/- 0.73 nM, Bmax 0.73 +/- 0.06 pmol/g wet weight and Hill coefficient 0.98 +/- 0.01. Specific binding of [3H]SP was displaced by SP greater than neurokinin A (NKA) greater than SP(3-11) approximately SP(1-9) greater than SP(1-7) approximately SP(1-4) approximately SP(1-6), with neurokinin B (NKB) and SP(1-3) very weak competitors and SP(5-11), SP(7-11) and SP(9-11) causing negligible inhibition (up to 10 microM). This potency order is quite distinct from that seen with binding to an NK1 site, a conclusion confirmed by the lack of BHSP binding. It appears that Lys3 and/or Pro4 are critical for binding, suggesting an anionic binding site. These data suggest the existence of an unusual binding site which may represent a novel SP receptor. This site appears to require the entire sequence of the SP molecule for full recognition.

L-baclofen-sensitive GABAB binding sites in the medial vestibular nucleus localized by immunocytochemistry

NASA Technical Reports Server (NTRS)

Holstein, G. R.; Martinelli, G. P.; Cohen, B.

1992-01-01

L-Baclofen-sensitive GABAB binding sites in the medial vestibular nucleus (MVN) were identified immunocytochemically and visualized ultrastructurally in L-baclofen-preinjected rats and monkeys, using a mouse monoclonal antibody with specificity for the p-chlorophenyl moiety of baclofen. Saline-preinjected animals showed no immunostain. In drug-injected animals, there was evidence for both pre- and postsynaptic GABAergic inhibition in MVN mediated by GABAB receptors. These neural elements could be utilized in control of velocity storage in the vestibulo-ocular reflex.

Crystal Structure of Heart 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase (PFKFB2) and the Inhibitory Influence of Citrate on Substrate Binding

PubMed Central

Crochet, Robert B.; Kim, Jeong-Do; Lee, Herie; Yim, Young-Sun; Kim, Song-Gun; Neau, David; Lee, Yong-Hwan

2016-01-01

The heart-specific isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8Å, respectively. Citrate, a TCA cycle intermediate and well-known inhibitor of PFKFB2, co-crystallized in the 2-kinase domains of both orthologues, occupying the fructose-6-phosphate binding-site and extending into the γ-phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ-phosphate site by citrate proved highly consequential to the binding of co-complexed ATP analogues. The bovine structure, which co-crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co-complexed with AMPPNP, which, unlike ADP, contains a γ-phosphate. The presence of this γ-phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding-pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate’s inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate’s negative feed-back loop of the glycolytic pathway via PFKFB2. PMID:27802586

Expression of receptors for atrial natriuretic peptide on the murine bone marrow-derived stromal cells.

PubMed

Agui, T; Yamada, T; Legros, G; Nakajima, T; Clark, M; Peschel, C; Matsumoto, K

1992-05-01

Atrial natriuretic peptide (ANP) receptors were identified on both murine bone marrow-derived stromal cell lines A-3 and ALC and primary cultured cells using [125I]ANP binding assays and Northern blot analyses. The binding of [125I] ANP to the stromal cells was rapid, saturable, and of high affinity. The dissociation constants between ANP and its receptors on these cells showed no difference among cell types, while maximal binding capacity values were different among cell types. Competitive inhibition of [125I]ANP binding with C-atrial natriuretic factor, specific for ANP clearance receptor (ANPR-C), revealed that most of [125I]ANP-binding sites corresponded to ANPR-C. Northern blotting data corroborated that bone marrow-derived stromal cells expressed ANPR-C. However, in ALC cells, ANP biological receptors (either ANPR-A or ANPR-B), the mol wt of which is approximately 130K, were detected, and cGMP was accumulated after stimulation with ANP. On the other hand, in another stromal cell clone, A-3 cells, the expression of biological receptor was not detected in the affinity cross-linking and competitive inhibition experiments using [125I]ANP. However, A-3 cells accumulated cGMP by responding to ANPR-B-specific ligand, C-type natriuretic peptide. These results suggest that ALC cells equally express ANPR-A and ANPR-B, while A-3 cells express ANPR-B dominantly. Although the physiological roles of these receptors in the bone marrow is still not resolved, ANP is expected to play a role in the regulation of stromal cell functions in bone marrow.

Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

PubMed

Kawamoto, Norio; Kamemura, Norio; Kido, Hiroshi; Fukao, Toshiyuki

2017-06-01

Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Long noncoding RNA FTX inhibits hepatocellular carcinoma proliferation and metastasis by binding MCM2 and miR-374a.

PubMed

Liu, F; Yuan, J-H; Huang, J-F; Yang, F; Wang, T-T; Ma, J-Z; Zhang, L; Zhou, C-C; Wang, F; Yu, J; Zhou, W-P; Sun, S-H

2016-10-13

It has long been known that males are more susceptible than females to hepatocellular carcinoma (HCC), but the reason remains elusive. In this study, we investigated the expression and function of the long noncoding RNA FTX (lnc-FTX), an X-inactive-specific transcript (XIST) regulator transcribed from the X chromosome inactivation center, in both HCC and HCC gender disparity. lnc-FTX is expressed at higher levels in female livers than in male livers and is significantly downregulated in HCC tissues compared with normal liver tissues. Patients with higher lnc-FTX expression exhibited longer survival, suggesting that lnc-FTX is a useful prognostic factor for HCC patients. lnc-FTX inhibits HCC cell growth and metastasis both in vitro and in vivo. Mechanistically, lnc-FTX represses Wnt/β-catenin signaling activity by competitively sponging miR-374a and inhibits HCC cell epithelial-mesenchymal transition and invasion. In addition, lnc-FTX binds to the DNA replication licensing factor MCM2, thereby impeding DNA replication and inhibiting proliferation in HCC cells. In conclusion, these findings suggest that lnc-FTX may act as a tumor suppressor in HCC through physically binding miR-374a and MCM2. It may also be one of the reasons for HCC gender disparity and may potentially contribute to HCC treatment.

Detection of Necroptosis in Ligand-Mediated and Hypoxia-Induced Injury of Hepatocytes Using a Novel Optic Probe Detecting Receptor-Interacting Protein (RIP)1/RIP3 Binding.

PubMed

Haga, Sanae; Kanno, Akira; Ozawa, Takeaki; Morita, Naoki; Asano, Mami; Ozaki, Michitaka

2017-07-21

Liver injury is often observed in various pathological conditions including posthepatectomy state and cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell death. In order to study liver/hepatocyte injury by necrotic type of cell death, we studied signal-regulated necrosis (necroptosis) by newly developing an optic probe detecting receptor-interacting protein (RIP)1/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 cells, TNF-a/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by z-VAD-fmk (zVAD), a caspase-specific inhibitor. T/C/zVADinduced RIP1/3-binding was inhibited by necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when apoptotic pathway is inhibited/unavailable. FasL also induced cell death which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis other than apoptosis. FasL activated caspase-3 and, similarly, induced RIP1/3-binding when caspases were inactivated. Interestingly, FasL-induced RIP1/3 binding was significantly suppressed by the antioxidants, Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H₂O₂, by itself, induced RIP1/3 binding that was suppressed by Nec-1, but not by zVAD. Hypoxia induced RIP1/3 binding after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/reoxygenation (H/R) was also improved by Nec-1. Similar to H₂O₂, H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligand induced and/or redox-mediated necroptosis. These data indicate that ROS induce necroptosis, and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional caspase-dependent pathway. In conclusion, necroptosis is potentially involved in liver/hepatocyte injury induced by oxidative stress and FasL, other than apoptosis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steiner, B.; Cousot, D.; Trzeciak, A.

The platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a member of the integrin receptor family that recognizes adhesive proteins containing the Arg-Gly-Asp (RGD) sequence. In the present study the binding characteristics of the synthetic hexapeptide Tyr-Asn-Arg-Gly-Asp-Ser (YNRGDS, a sequence present in the fibrinogen alpha-chain at position 570-575) to purified GP IIb-IIIa were determined by equilibrium dialysis. The binding of 125I-YNRGDS to GP IIb-IIIa was specific, saturable, and reversible. The apparent dissociation constant was 1.0 +/- 0.2 microM, and the maximal binding capacity was 0.92 +/- 0.02 mol of 125I-YNRGDS/mol of GP IIb-IIIa, indicating that GP IIb-IIIa contains a single bindingmore » site for RGD peptides. The binding of 125I-YNRGDS to purified GP IIb-IIIa showed many of the characteristics of fibrinogen binding to activated platelets: the binding was inhibited by fibrinogen, by the monoclonal antibody A2A9, and by the dodecapeptide from the C terminus of the fibrinogen gamma-chain. In addition, the binding of 125I-YNRGDS to GP IIb-IIIa was divalent cation-dependent. Our data suggest that two divalent cation binding sites must be occupied for YNRGDS to bind: one site is specific for calcium and is saturated at 1 microM free Ca2+, whereas the other site is less specific and reaches saturation at millimolar concentrations of either Ca2+ or Mg2+. The results of the present study support the hypothesis that the RGD domains within the adhesive proteins are responsible for their binding to GP IIb-IIIa.« less

Lactobacillus buchneri S-layer as carrier for an Ara h 2-derived peptide for peanut allergen-specific immunotherapy.

PubMed

Anzengruber, Julia; Bublin, Merima; Bönisch, Eva; Janesch, Bettina; Tscheppe, Angelika; Braun, Matthias L; Varga, Eva-Maria; Hafner, Christine; Breiteneder, Heimo; Schäffer, Christina

2017-05-01

Peanut allergy is an IgE-mediated severe hypersensitivity disorder. The lack of a treatment of this potentially fatal allergy has led to intensive research on vaccine development. Here, we describe the design and initial characterization of a carrier-bound peptide derived from the most potent peanut allergen, Ara h 2, as a candidate vaccine. Based on the adjuvant capability of bacterial surface (S-) layers, a fusion protein of the S-layer protein SlpB from Lactobacillus buchneri CD034 and the Ara h 2-derived peptide AH3a42 was produced. This peptide comprised immunodominant B-cell epitopes as well as one T cell epitope. The fusion protein SlpB-AH3a42 was expressed in E. coli, purified, and tested for its IgE binding capacity as well as for its ability to activate sensitized rat basophil leukemia (RBL) cells. The capacity of Ara h 2-specific IgG rabbit-antibodies raised against SlpB-AH3a42 or Ara h 2 to inhibit IgE-binding was determined by ELISA inhibition assays using sera of peanut allergic patients sensitized to Ara h 2. IgE specific to the SlpB-AH3a42 fusion protein was detected in 69% (25 of 36) of the sera. Despite the recognition by IgE, the SlpB-AH3a42 fusion protein was unable to induce β-hexosaminidase release from sensitized RBL cells at concentrations up to 100ng per ml. The inhibition of IgE-binding to the natural allergen observed after pre-incubation of the 20 sera with rabbit anti-SlpB-AH3a42 IgG was more than 30% for four sera, more than 20% for eight sera, and below 10% for eight sera. In comparison, anti-Ara h 2 rabbit IgG antibodies inhibited binding to Ara h 2 by 48% ±13.5%. Our data provide evidence for the feasibility of this novel approach towards the development of a peanut allergen peptide-based carrier-bound vaccine. Our experiments further indicate that more than one allergen-peptide will be needed to induce a broader protection of patients allergic to Ara h 2. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Distinct mechanisms for N-acetylcysteine inhibition of cytokine-induced E-selectin and VCAM-1 expression.

PubMed

Faruqi, R M; Poptic, E J; Faruqi, T R; De La Motte, C; DiCorleto, P E

1997-08-01

We have examined the effects of N-acetyl-L-cysteine (NAC), a well-characterized, thiol-containing antioxidant, on agonist-induced monocytic cell adhesion to endothelial cells (EC). NAC inhibited interleukin-1 (IL-1 beta)-induced, but not basal, adhesion with 50% inhibition at approximately 20 mM. Monocytic cell adhesion to EC in response to tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA) was similarly inhibited by NAC. Unlike published studies with pyrrolidinedithiocarbamate, which specifically inhibited vascular cell adhesion molecule 1 (VCAM-1), NAC inhibited IL-1 beta-induced mRNA and cell surface expression of both E-selectin and VCAM-1. NAC had no effect on the half-life of E-selectin or VCAM-1 mRNA. Although NAC reduced nuclear factor-kappa B (NF-kappa B) activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding to the consensus NF-kappa B binding sites of the VCAM-1 gene (VCAM-NF-kappa B), the antioxidant had no appreciable effect when an oligomer corresponding to the consensus NF-kappa B binding site of the E-selectin gene (E-selectin-NF-kappa B) was used. Because NF-kappa B has been reported to be redox sensitive, we studied the effects of NAC on the EC redox environment. NAC caused an expected dramatic increase in the reduced glutathione (GSH) levels in EC. In vitro studies demonstrated that whereas the binding affinity of NF-kappa B to the VCAM-NF-kappa B oligomer peaked at a GSH-to-oxidized glutathione (GSSG) ratio of approximately 200 and decreased at higher ratios, the binding to the E-selectin-NF-kappa B oligomer appeared relatively unaffected even at ratios > 400, i.e., those achieved in EC treated with 40 mM NAC. These results suggest that NF-kappa B binding to its consensus sequences in the VCAM-1 and E-selectin gene exhibits marked differences in redox sensitivity, allowing for differential gene expression regulated by the same transcription factor. Our data also demonstrate that NAC increases the GSH-to-GSSG ratio within the EC suggesting one possible mechanism through which this antioxidant inhibits agonist-induced monocyte adhesion to EC.

Human milk glycoconjugates that inhibit pathogens.

PubMed

Newburg, D S

1999-02-01

Breast-fed infants have lower incidence of diarrhea, respiratory disease, and otitis media. The protection by human milk has long been attributed to the presence of secretory IgA. However, human milk contains large numbers and amounts of complex carbohydrates, including glycoproteins, glycolipids, glycosaminoglycans, mucins, and especially oligosaccharides. The oligosaccharides comprise the third most abundant solid constituent of human milk, and contain a myriad of structures. Complex carbohydrate moieties of glycoconjugates and oligosaccharides are synthesized by the many glycosyltransferases in the mammary gland; those with homology to cell surface glycoconjugate pathogen receptors may inhibit pathogen binding, thereby protecting the nursing infant. Several examples are reviewed: A fucosyloligosaccharide inhibits the diarrheagenic effect of stable toxin of Escherichia coli. A different fucosyloligosaccharide inhibits infection by Campylobacter jejuni. Binding of Streptococcus pneumoniae and of enteropathogenic E. coli to their respective receptors is inhibited by human milk oligosaccharides. The 46-kD glycoprotein, lactadherin, inhibits rotavirus binding and infectivity. Low levels of lactadherin in human milk are associated with a higher incidence of symptomatic rotavirus in breast-fed infants. A mannosylated glycopeptide inhibits binding by enterohemorrhagic E. coli. A glycosaminoglycan inhibits binding of gp120 to CD4, the first step in HIV infection. Human milk mucin inhibits binding by S-fimbriated E. coli. The ganglioside, GM1, reduces diarrhea production by cholera toxin and labile toxin of E. coli. The neutral glycosphingolipid, Gb3, binds to Shigatoxin. Thus, many complex carbohydrates of human milk may be novel antipathogenic agents, and the milk glycoconjugates and oligosaccharides may be a major source of protection for breastfeeding infants.

Two-phase positive inotropic effects of ouabain and the presence of multiple classes of ouabain binding sites in the ferret heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ng, Y.C.; Akera, T.

1986-03-05

Characteristics of more than one class of ouabain receptors which appear to exist in ferret heart were examined. In isolated papillary muscle, 1 to 30 nM ouabain produced a positive inotropic effect in the presence of 5 ..mu..M propranolol and 2 ..mu..M phentolamine. Higher concentrations of ouabain (0.1 to 10 ..mu..M) produced an additional and prominent inotropic effect. In partially purified Na, K-ATPase, ouabain caused a monophasic inhibition; however, the concentration-inhibition curve spanned over 5 log units, indicating that ouabain is interacting with more than a single class of the enzyme. Scatchard analysis of specific /sup 3/H-ouabain binding revealed approximatelymore » equal abundance of high and low affinity binding sites. The K/sub D/ value for high affinity sites was approximately 20 nM whereas that for low affinity sites was about 45 times higher. When phosphoenzyme was formed in the presence of (..gamma..-/sup 32/P)-ATP, Mg/sup 2 +/ and Na/sup +/ and subjected to SDS gel electrophoresis, two distinct K/sup +/-sensitive bands with about 100,000 dalton molecular weight were detected. Molecular weight difference between these two bands was approximately 2500 dalton. Phosphorylation of either band was abolished by 1 ..mu..M ouabain suggesting that both bands may correspond to the high-affinity binding sites. These results indicate that high and low affinity ouabain binding sites exists in approximately equal abundance in the ferret heart, and that binding of ouabain to these sites cases Na,K-ATPase inhibition and the positive inotropic effect.« less

Presynaptic imidazoline receptors and non-adrenoceptor[3H]-idazoxan binding sites in human cardiovascular tissues

PubMed Central

Molderings, G J; Likungu, J; Jakschik, J; Göthert, M

1997-01-01

In segments of human right atrial appendages and pulmonary arteries preincubated with [3H]-noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, the involvement of imidazoline receptors in the modulation of [3H]-noradrenaline release was investigated. In human atrial appendages, the guanidines aganodine and DTG (1,3-di(2-tolyl)guanidine) which activate presynaptic imidazoline receptors, inhibited electrically-evoked [3H]-noradrenaline release. The inhibition was not affected by blockade of α2-adrenoceptors with 1 μM rauwolscine, but antagonized by extremely high concentrations of this drug (10 and/or 30 μM; apparent pA2 against aganodine and DTG: 5.55 and 5.21, respectively). In the presence of 1 μM rauwolscine, [3H]-noradrenaline release in human atrial appendages was also inhibited by the imidazolines idazoxan and cirazoline, but not by agmatine and noradrenaline. The inhibitory effects of 100 μM idazoxan and 30 μM cirazoline were abolished by 30 μM rauwolscine. In the atrial appendages, the rank order of potency of all guanidines and imidazolines for their inhibitory effect on electrically-evoked [3H]-noradrenaline release in the presence of 1 μM rauwolscine was: aganodine⩾BDF 6143 [4-chloro-2-(2-imidazolin-2-yl-amino)-isoindoline]>DTG⩾clonidine>cirazoline>idazoxan (BDF 6143 and clonidine were previously studied under identical conditions). This potency order corresponded to that previously determined at the presynaptic imidazoline receptors in the rabbit aorta. When, in the experiments in the human pulmonary artery, rauwolscine was absent from the superfusion fluid, the concentration-response curve for BDF 6143 (a mixed α2-adrenoceptor antagonist/imidazoline receptor agonist) for its facilitatory effect on electrically-evoked [3H]-noradrenaline release was bell-shaped. In the presence of 1 μM rauwolscine, BDF 6143 and cirazoline concentration-dependently inhibited the evoked [3H]-noradrenaline release. In human atrial appendages, non-adrenoceptor [3H]-idazoxan binding sites were identified and characterized. The binding of [3H]-idazoxan was specific, reversible, saturable and of high affinity (KD: 25.5 nM). The specific binding of [3H]-idazoxan (defined by cirazoline 0.1 mM) to membranes of human atrial appendages was concentration-dependently inhibited by several imidazolines and guanidines, but not by rauwolscine and agmatine. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases for the only detectable site; cirazoline=idazoxan>BDF 6143>DTG⩾clonidine) is compatible with the pharmacological properties of I2-imidazoline binding sites, but is clearly different from the rank order of potency for inhibiting evoked noradrenaline release from sympathetic nerves in the same tissue. It is concluded that noradrenaline release in the human atrium and, less well established, in the pulmonary artery is inhibited via presynaptic imidazoline receptors. These presynaptic imidazoline receptors appear to be related to those previously characterized in rabbit aorta and pulmonary artery, but differ clearly from I1 and I2 imidazoline binding sites. PMID:9298527

Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

PubMed Central

Ll, Juan Modole; Cabrer, Bartolomé; Parmeggiani, Andrea; Azquez, David V

1971-01-01

Siomycin, a peptide antibiotic that interacts with the 50S ribosomal subunit and inhibits binding of factor G, is shown also to inhibit binding of aminoacyl-tRNA; however, it does not impair binding of fMet-tRNA and completion of the initiation complex. Moreover, unlike other inhibitors of aminoacyl-tRNA binding (tetracycline, sparsomycin, and streptogramin A), siomycin completely abolishes the GTPase activity associated with the binding of aminoacyl-tRNA catalyzed by factor Tu. A single-site interaction of siomycin appears to be responsible for its effect on both the binding of the aminoacyl-tRNA-Tu-GTP complex and that of factor G. PMID:4331558

An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis

DOE PAGES

Kariolis, Mihalis S.; Miao, Yu Rebecca; Jones, Douglas S.; ...

2014-09-21

Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl ‘decoy receptor’ that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high affinity Axl variant caused structural alterations in side chains across the Gas6/Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc-fusion, the engineered decoy receptor bound tomore » Gas6 with femtomolar affinity, an 80-fold improvement compared to the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Additionally, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.« less

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells.

PubMed

Tahara, A; Tsukada, J; Tomura, Y; Wada, K i; Kusayama, T; Ishii, N; Yatsu, T; Uchida, W; Tanaka, A

2000-01-01

[(3)H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [(3)H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (K(d)) of 0.76 nM and a maximum receptor density (B(max)) of 153 fmol mg(-1) protein. The Hill coefficient (n(H)) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [(3)H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [(3)H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu(1,6)]-oxytocin>AVP= atosiban>d(CH(2))(5)Tyr(Me)AVP>[Thr(4),Gly(7)]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca(2+)](i) increase and hyperplasia. In contrast, the V(1A) receptor selective antagonist, SR 49059, and the V(2) receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca(2+)](i) increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca(2+)](i) increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [(3)H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca(2+)](i) increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. British Journal of Pharmacology (2000) 129, 131 - 139

Inhibition of benzodiazepine binding in vitro by amentoflavone, a constituent of various species of Hypericum.

PubMed

Baureithel, K H; Büter, K B; Engesser, A; Burkard, W; Schaffner, W

1997-06-01

Flower extracts of Hypericum perforatum, Hypericum hirsutum, Hypericum patulum and Hypericum olympicum efficiently inhibited binding of [3H]flumazenil to rat brain benzodiazepine binding sites of the GABAA-receptor in vitro with IC50 values of 6.83, 6.97, 13.2 and 6.14 micrograms/ml, respectively. Single constituents of the extracts like hypericin, the flavones quercetin and luteolin, the glycosylated flavonoides rutin, hyperoside and quercitrin and the biflavone 13, II8-biapigenin did not inhibit binding up to concentrations of 1 microM. In contrast, amentoflavone revealed an IC50 = 14.9 +/- 1.9 nM on benzodiazepine binding in vitro. Comparative HPLC analyses of hypericin and amentoflavone in extracts of different Hypericum species revealed a possible correlation between the amentoflavone concentration and the inhibition of flumazenil binding. For hypericin no such correlation was observed. Our experimental data demonstrate that amentoflavone, in contrast to hypericin, presents a very active compound with regard to the inhibition of [3H]-flumazenil binding in vitro and thus might be involved in the antidepressant effects of Hypericum perforatum extracts.

Aminopeptidase A is a functional target in angiogenic blood vessels.

PubMed

Marchiò, Serena; Lahdenranta, Johanna; Schlingemann, Reinier O; Valdembri, Donatella; Wesseling, Pieter; Arap, Marco A; Hajitou, Amin; Ozawa, Michael G; Trepel, Martin; Giordano, Ricardo J; Nanus, David M; Dijkman, Henri B P M; Oosterwijk, Egbert; Sidman, Richard L; Cooper, Max D; Bussolino, Federico; Pasqualini, Renata; Arap, Wadih

2004-02-01

We show that a membrane-associated protease, aminopeptidase A (APA), is upregulated and enzymatically active in blood vessels of human tumors. To gain mechanistic insight, we evaluated angiogenesis in APA null mice. We found that, although these mice develop normally, they fail to mount the expected angiogenic response to hypoxia or growth factors. We then isolated peptide inhibitors of APA from a peptide library and show that they specifically bind to and inhibit APA, suppress migration and proliferation of endothelial cells, inhibit angiogenesis, and home to tumor blood vessels. Finally, we successfully treated tumor-bearing mice with APA binding peptides or anti-APA blocking monoclonal antibodies. These data show that APA is a regulator of blood vessel formation, and can serve as a functional vascular target.

Boric acid reversibly inhibits the second step of pre-mRNA splicing.

PubMed

Shomron, Noam; Ast, Gil

2003-09-25

Several approaches have been used to identify the factors involved in mRNA splicing. None of them, however, comprises a straightforward reversible method for inhibiting the second step of splicing using an external reagent other than a chelator. This investigation demonstrates that the addition of boric acid to an in vitro pre-mRNA splicing reaction causes a dose-dependent reversible inhibition effect on the second step of splicing. The mechanism of action does not involve chelation of several metal ions; hindrance of 3' splice-site; or binding to hSlu7. This study presents a novel method for specific reversible inhibition of the second step of pre-mRNA splicing.

Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein.

PubMed

Zhang, Zhen; Palzkill, Timothy

2003-11-14

The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.

Aptamer-Conjugated Calcium Phosphate Nanoparticles for Reducing Diabetes Risk via Retinol Binding Protein 4 Inhibition.

PubMed

Torabi, Raheleh; Ghourchian, Hedayatollah; Amanlou, Massoud; Pasalar, Parvin

2017-06-01

Inhibition of the binding of retinol to its carrier, retinol binding protein 4, is a new strategy for treating type 2 diabetes; for this purpose, we have provided an aptamer-functionalized multishell calcium phosphate nanoparticle. First, calcium phosphate nanoparticles were synthesized and conjugated to the aptamer. The cytotoxicity of nanoparticles releases the process of aptamer from nanoparticles and their inhibition function of binding retinol to retinol binding protein 4. After synthesizing and characterizing the multishell calcium phosphate nanoparticles and observing the noncytotoxicity of conjugate, the optimum time (48 hours) and the pH (7.4) for releasing the aptamer from the nanoparticles was determined. The half-maximum inhibitory concentration (IC 50 ) value for inhibition of retinol binding to retinol binding protein 4 was 210 femtomolar (fmol). The results revealed that the aptamer could prevent connection between retinol and retinol binding protein 4 at a very low IC 50 value (210 fmol) compared to other reported inhibitors. It seems that this aptamer could be used as an efficient candidate not only for decreasing the insulin resistance in type 2 diabetes, but also for inhibiting the other retinol binding protein 4-related diseases. Copyright © 2017 Diabetes Canada. Published by Elsevier Inc. All rights reserved.

Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Seong K., E-mail: skim1@lsuhsc.edu; Kim, Seongman; Dai Gan

2011-09-01

The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% ofmore » control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: > We examine the functional domains of IR2P that mediates negative regulation. > IR2P inhibits at the transcriptional level. > DNA-binding mutant or TFIIB-binding mutant fails to inhibit. > C-terminal aa 707 to 1116 are required for full inhibition. > Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.« less

Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands

PubMed Central

Granovsky, Alexey E.; Clark, Mathew M.; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R.; Koide, Shohei

2010-01-01

Background Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. Methods/Findings In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. Conclusions/Significance This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential. PMID:20463977

Mechanism for CARMIL Protein Inhibition of Heterodimeric Actin-capping Protein*

PubMed Central

Kim, Taekyung; Ravilious, Geoffrey E.; Sept, David; Cooper, John A.

2012-01-01

Capping protein (CP) controls the polymerization of actin filaments by capping their barbed ends. In lamellipodia, CP dissociates from the actin cytoskeleton rapidly, suggesting the possible existence of an uncapping factor, for which the protein CARMIL (capping protein, Arp2/3 and myosin-I linker) is a candidate. CARMIL binds to CP via two motifs. One, the CP interaction (CPI) motif, is found in a number of unrelated proteins; the other motif is unique to CARMILs, the CARMIL-specific interaction motif. A 115-aa CARMIL fragment of CARMIL with both motifs, termed the CP-binding region (CBR), binds to CP with high affinity, inhibits capping, and causes uncapping. We wanted to understand the structural basis for this function. We used a collection of mutants affecting the actin-binding surface of CP to test the possibility of a steric-blocking model, which remained open because a region of CBR was not resolved in the CBR/CP co-crystal structure. The CP actin-binding mutants bound CBR normally. In addition, a CBR mutant with all residues of the unresolved region changed showed nearly normal binding to CP. Having ruled out a steric blocking model, we tested an allosteric model with molecular dynamics. We found that CBR binding induces changes in the conformation of the actin-binding surface of CP. In addition, ∼30-aa truncations on the actin-binding surface of CP decreased the affinity of CBR for CP. Thus, CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP. PMID:22411988

Pharmacological activities of Vitex agnus-castus extracts in vitro.

PubMed

Meier, B; Berger, D; Hoberg, E; Sticher, O; Schaffner, W

2000-10-01

The pharmacological effects of ethanolic Vitex agnus-castus fruit-extracts (especially Ze 440) and various extract fractions of different polarities were evaluated both by radioligand binding studies and by superfusion experiments. A relative potent binding inhibition was observed for dopamine D2 and opioid (micro and kappa subtype) receptors with IC50 values of the native extract between 20 and 70 mg/mL. Binding, neither to the histamine H1, benzodiazepine and OFQ receptor, nor to the binding-site of the serotonin (5-HT) transporter, was significantly inhibited. The lipophilic fractions contained the diterpenes rotun-difuran and 6beta,7beta-diacetoxy-13-hydroxy-labda-8,14-dien . They exhibited inhibitory actions on dopamine D2 receptor binding. While binding inhibition to mu and kappa opioid receptors was most pronounced in lipophilic fractions, binding to delta opioid receptors was inhibited mainly by a aqueous fraction. Standardised Ze 440 extracts of different batches were of constant pharmacological quality according to their potential to inhibit the binding to D2 receptors. In superfusion experiments, the aqueous fraction of a methanolic extract inhibited the release of acetylcholine in a concentration-dependent manner. In addition, the potent D2 receptor antagonist spiperone antagonised the effect of the extract suggesting a dopaminergic action mediated by D2 receptor activation. Our results indicate a dopaminergic effect of Vitex agnus-castus extracts and suggest additional pharmacological actions via opioid receptors.

The rhizotoxicity of metal cations is related to their strength of binding to hard ligands.

PubMed

Kopittke, Peter M; Menzies, Neal W; Wang, Peng; McKenna, Brigid A; Wehr, J Bernhard; Lombi, Enzo; Kinraide, Thomas B; Blamey, F Pax C

2014-02-01

Mechanisms whereby metal cations are toxic to plant roots remain largely unknown. Aluminum, for example, has been recognized as rhizotoxic for approximately 100 yr, but there is no consensus on its mode of action. The authors contend that the primary mechanism of rhizotoxicity of many metal cations is nonspecific and that the magnitude of toxic effects is positively related to the strength with which they bind to hard ligands, especially carboxylate ligands of the cell-wall pectic matrix. Specifically, the authors propose that metal cations have a common toxic mechanism through inhibiting the controlled relaxation of the cell wall as required for elongation. Metal cations such as Al(3+) and Hg(2+), which bind strongly to hard ligands, are toxic at relatively low concentrations because they bind strongly to the walls of cells in the rhizodermis and outer cortex of the root elongation zone with little movement into the inner tissues. In contrast, metal cations such as Ca(2+), Na(+), Mn(2+), and Zn(2+) , which bind weakly to hard ligands, bind only weakly to the cell wall and move farther into the root cylinder. Only at high concentrations is their weak binding sufficient to inhibit the relaxation of the cell wall. Finally, different mechanisms would explain why certain metal cations (for example, Tl(+), Ag(+), Cs(+), and Cu(2+)) are sometimes more toxic than expected through binding to hard ligands. The data presented in the present study demonstrate the importance of strength of binding to hard ligands in influencing a range of important physiological processes within roots through nonspecific mechanisms. © 2013 SETAC.

Heterogeneous Nuclear Ribonucleoprotein (hnRNP) E1 Binds to hnRNP A2 and Inhibits Translation of A2 Response Element mRNAs

PubMed Central

Kosturko, Linda D.; Maggipinto, Michael J.; Korza, George; Lee, Joo Won; Carson, John H.

2006-01-01

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport. PMID:16775011

Sulfated Low Molecular Weight Lignins, Allosteric Inhibitors of Coagulation Proteinases via the Heparin Binding Site, Significantly Alter the Active Site of Thrombin and Factor Xa Compared to Heparin

PubMed Central

Henry, Brian L.; Desai, Umesh R.

2014-01-01

Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. PMID:25242245

Sulfated low molecular weight lignins, allosteric inhibitors of coagulation proteinases via the heparin binding site, significantly alter the active site of thrombin and factor xa compared to heparin.

PubMed

Henry, Brian L; Desai, Umesh R

2014-11-01

Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. Copyright © 2014 Elsevier Ltd. All rights reserved.

Feline immunodeficiency virus envelope glycoprotein mediates apoptosis in activated PBMC by a mechanism dependent on gp41 function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garg, Himanshu; Joshi, Anjali; Tompkins, Wayne A.

2004-12-20

Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model.more » Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function.« less

beta. -Adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation

DOE Office of Scientific and Technical Information (OSTI.GOV)

van Koppen, C.J.; Hermanussen, M.W.; Verrijp, K.N.

1987-06-29

Specific binding of (/sup 125/I)-(-)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (K/sub d/ = 5.3 +/- 0.9 pmol/l and R/sub T/ = 78 +/- 7 fmol/g tissue). The ..beta../sub 1/-selective antagonists atenolol and LK 203-030 inhibited specific (/sup 125/I)-(-)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous BETA/sub 2/-adrenoceptor population. In one subject using LK 203-030 a small ..beta../sub 1/-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pK/sub H/-more » and pK/sub L/-values for the high and low affinity sites were 8.0 +/- 0.2 and 5.9 +/- 0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD/sub 2/-value of 6.63 +/- 0.19. 32 references, 4 figures, 2 tables.« less

Zinc chelation with hydroxamate in histone deacetylases modulated by water access to the linker binding channel.

PubMed

Wu, Ruibo; Lu, Zhenyu; Cao, Zexing; Zhang, Yingkai

2011-04-27

It is of significant biological interest and medical importance to develop class- and isoform-selective histone deacetylase (HDAC) modulators. The impact of the linker component on HDAC inhibition specificity has been revealed but is not understood. Using Born-Oppenheimer ab initio QM/MM MD simulations, a state-of-the-art approach to simulating metallo-enzymes, we have found that the hydroxamic acid remains to be protonated upon its binding to HDAC8, and thus disproved the mechanistic hypothesis that the distinct zinc-hydroxamate chelation modes between two HDAC subclasses come from different protonation states of the hydroxamic acid. Instead, our simulations suggest a novel mechanism in which the chelation mode of hydroxamate with the zinc ion in HDACs is modulated by water access to the linker binding channel. This new insight into the interplay between the linker binding and the zinc chelation emphasizes its importance and gives guidance regarding linker design for the development of new class-IIa-specific HDAC inhibitors.

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Pro-Convertase

PubMed Central

Garcia, Brandon L.; Ramyar, Kasra X.; Keightley, Andrew; Ruyken, Maartje; Syriga, Maria; Sfyroera, Georgia; Weber, Alexander B.; Zolkiewski, Michal; Ricklin, Daniel; Lambris, John D.; Rooijakkers, Suzan H.M.; Geisbrecht, Brian V.

2014-01-01

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. While the majority of staphylococcal complement inhibitors act on the alternative pathway (AP) to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical (CP) and lectin (LP) pathways. We screened a collection of recombinant, secreted staphylococcal proteins to determine if S. aureus produces other molecules that inhibit either the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 pro-convertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits the two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion. PMID:25381436

The C-terminal heavy-chain domain of botulinum neurotoxin a is not the only site that binds neurons, as the N-terminal heavy-chain domain also plays a very active role in toxin-cell binding and interactions.

PubMed

Ayyar, B Vijayalakshmi; Aoki, K Roger; Atassi, M Zouhair

2015-04-01

Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729-845). HN729-845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729-845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729-845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Purification and characterization of Lep d I, a major allergen from the mite Lepidoglyphus destructor.

PubMed

Ventas, P; Carreira, J; Polo, F

1992-04-01

A major allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been purified by affinity chromatography using an anti-Lep d I monoclonal antibody. The purity of the protein obtained by this procedure was assessed by reverse-phase HPLC. Lep d I displayed a molecular weight of 14 kD on SDS-PAGE under non-reducing conditions, and 16 kD in the presence of a reducing agent. Analytical IEF revealed a little charge microheterogeneity, showing three bands with pIs 7.6-7.8. Purified Lep d I retained IgE-binding ability, as proved by immunoblotting experiments after SDS-PAGE and RAST with individual sera from L. destructor-sensitive patients. Results from the latter technique demonstrated that 87% of L. destructor-allergic patients had specific IgE to Lep d I, and a good correlation between IgE reactivity with L. destructor extract and Lep d I was found. In addition, RAST inhibition experiments showed that IgE-binding sites on Lep d I are major L. destructor-allergenic determinants, since Lep d I could inhibit up to 75% the binding of specific IgE to L. destructor extract; on the other hand, Lep d I did not cross-react with D. pteronyssinus allergens.

Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site

PubMed Central

Sage, Jay M.; Cura, Anthony J.; Lloyd, Kenneth P.

2015-01-01

Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites—the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis. PMID:25715702

A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences

PubMed Central

Nagano, Yukio; Furuhashi, Hirofumi; Inaba, Takehito; Sasaki, Yukiko

2001-01-01

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x2-H-x11-C-x2-C-x(4–5)-C-x2-C-x(3–7)-H-x2-H and C-x2-C-x(10–11)-C-x3-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with 65ZnCl2 showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression. PMID:11600698

Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site.

PubMed

Matsumoto, Yuki; Shindo, Yosuke; Takakusagi, Yoichi; Takakusagi, Kaori; Tsukuda, Senko; Kusayanagi, Tomoe; Sato, Hitoshi; Kawabe, Takumi; Sugawara, Fumio; Sakaguchi, Kengo

2011-12-01

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix. Copyright © 2011 Elsevier Ltd. All rights reserved.

Immunization with Hypoallergens of Shrimp Allergen Tropomyosin Inhibits Shrimp Tropomyosin Specific IgE Reactivity

PubMed Central

Wai, Christine Y. Y.; Leung, Nicki Y. H.; Ho, Marco H. K.; Gershwin, Laurel J.; Shu, Shang An; Leung, Patrick S. C.; Chu, Ka Hou

2014-01-01

Designer proteins deprived of its IgE-binding reactivity are being sought as a regimen for allergen-specific immunotherapy. Although shrimp tropomyosin (Met e 1) has long been identified as the major shellfish allergen, no immunotherapy is currently available. In this study, we aim at identifying the Met e 1 IgE epitopes for construction of hypoallergens and to determine the IgE inhibitory capacity of the hypoallergens. IgE-binding epitopes were defined by three online computational models, ELISA and dot-blot using sera from shrimp allergy patients. Based on the epitope data, two hypoallergenic derivatives were constructed by site-directed mutagenesis (MEM49) and epitope deletion (MED171). Nine regions on Met e 1 were defined as the major IgE-binding epitopes. Both hypoallergens MEM49 and MED171 showed marked reduction in their in vitro reactivity towards IgE from shrimp allergy patients and Met e 1-sensitized mice, as well as considerable decrease in induction of mast cell degranulation as demonstrated in passive cutaneous anaphylaxis assay. Both hypoallergens were able to induce Met e 1-recognizing IgG antibodies in mice, specifically IgG2a antibodies, that strongly inhibited IgE from shrimp allergy subjects and Met e 1-sensitized mice from binding to Met e 1. These results indicate that the two designer hypoallergenic molecules MEM49 and MED171 exhibit desirable preclinical characteristics, including marked reduction in IgE reactivity and allergenicity, as well as ability to induce blocking IgG antibodies. This approach therefore offers promises for development of immunotherapeutic regimen for shrimp tropomyosin allergy. PMID:25365343

Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.

PubMed

Sha, Fern; Gencer, Emel Basak; Georgeon, Sandrine; Koide, Akiko; Yasui, Norihisa; Koide, Shohei; Hantschel, Oliver

2013-09-10

The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides.

PubMed

Kaconis, Yani; Kowalski, Ina; Howe, Jörg; Brauser, Annemarie; Richter, Walter; Razquin-Olazarán, Iosu; Iñigo-Pestaña, Melania; Garidel, Patrick; Rössle, Manfred; Martinez de Tejada, Guillermo; Gutsmann, Thomas; Brandenburg, Klaus

2011-06-08

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Overcoming Antigenic Diversity by Enhancing the Immunogenicity of Conserved Epitopes on the Malaria Vaccine Candidate Apical Membrane Antigen-1

PubMed Central

Dutta, Sheetij; Dlugosz, Lisa S.; Drew, Damien R.; Ge, Xiopeng; Ababacar, Diouf; Rovira, Yazmin I.; Moch, J. Kathleen; Shi, Meng; Long, Carole A.; Foley, Michael; Beeson, James G.; Anders, Robin F.; Miura, Kazutoyo; Haynes, J. David; Batchelor, Adrian H.

2013-01-01

Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes. PMID:24385910

A novel core 1 O-linked glycan-specific binding lectin from the fruiting body of Hericium erinaceus.

PubMed

Kim, Seonghun

2018-02-01

Mucin-type O-glycans are involved in biological functions on the cell surface as well as the glycoproteins and can also be used as specific carbohydrate biomarkers of many diseases. In this study, I purified a novel core 1 O-linked glycan specific lectin, Hericium erinaceus lecin (HeL), from the fruiting body of the mushroom Hericium erinaceus, which is known as the natural source for a sialic acid-binding lectin. Upon optimization of the purification conditions, a sequence of ion exchange, affinity, ion exchange, and size-exclusion chromatography resulted in the highest yield and best quality of lectin without protease activity. The resulting purified HeL is an apparent hexameric protein with a subunit molecular weight of 15kDa, and a pI of 4.3. In hemagglutination inhibition assay, the purified lectin was only inhibited by glycoproteins containing mucin-type O-glycans and reacted weakly with Galβ(1,3)GalNAc. Glycan array analyses showed that HeL specifically interacts with core 1 O-linked glycans as well as extended O-glycan structures containing sialylation or fucosylation. The glycan binding specificity of HeL is comparable to that of peanut agglutinin for detection of a broader range of extended core 1 O-glycan structures. Taken together, these results provide an efficient and optimized procedure for the purification of HeL from the fruiting body of the mushroom Hericium erinaceus. Moreover, HeL represents a powerful tool for analyzing core 1 and extended core 1 O- glycan structures in diagnosis assays. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibition of experimental ascending urinary tract infection by an epithelial cell-surface receptor analogue

NASA Astrophysics Data System (ADS)

Edén, C. Svanborg; Freter, R.; Hagberg, L.; Hull, R.; Hull, S.; Leffler, H.; Schoolnik, G.

1982-08-01

It has been shown that the establishment of urinary tract infection by Escherichia coli is dependent on attachment of the bacteria to epithelial cells1-4. The attachment involves specific epithelial cell receptors, which have been characterized as glycolipids5-10. Reversible binding to cell-surface mannosides may also be important4,11-13. This suggests an approach to the treatment of infections-that of blocking bacterial attachment with cell membrane receptor analogues. Using E. coli mutants lacking one or other of the two binding specificities (glycolipid and mannose), we show here that glycolipid analogues can block in vitro adhesion and in vivo urinary tract infection.

Specific interactions between mycobacterial FtsZ protein and curcumin derivatives: Molecular docking and ab initio molecular simulations

NASA Astrophysics Data System (ADS)

Fujimori, Mitsuki; Sogawa, Haruki; Ota, Shintaro; Karpov, Pavel; Shulga, Sergey; Blume, Yaroslav; Kurita, Noriyuki

2018-01-01

Filamentous temperature-sensitive Z (FtsZ) protein plays essential role in bacteria cell division, and its inhibition prevents Mycobacteria reproduction. Here we adopted curcumin derivatives as candidates of novel inhibitors and investigated their specific interactions with FtsZ, using ab initio molecular simulations based on protein-ligand docking, classical molecular mechanics and ab initio fragment molecular orbital (FMO) calculations. Based on FMO calculations, we specified the most preferable site of curcumin binding to FtsZ and highlighted the key amino acid residues for curcumin binding at an electronic level. The result will be useful for proposing novel inhibitors against FtsZ based on curcumin derivatives.

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

Site-Specific 64Cu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N3), Using Copper Free Click Chemistry.

PubMed

Jeppesen, Troels E; Kristensen, Lotte K; Nielsen, Carsten H; Petersen, Lars C; Kristensen, Jesper B; Behrens, Carsten; Madsen, Jacob; Kjaer, Andreas

2018-01-17

A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with 64 Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migration, and survival of cancer cells. First a single azide moiety was introduced in the active site of this 50 kDa protease. Then a NOTA moiety was introduced via a strain promoted azide-alkyne reaction and the corresponding conjugate was labeled with 64 Cu. Binding to TF and the stability was evaluated in vitro. TF targeting capability of the radiolabeled conjugate was tested in vivo by positron emission tomography (PET) imaging in pancreatic human xenograft cancer mouse models with various TF expressions. The conjugate showed good stability (>91% at 16 h), an immunoreactivity of 93.5%, and a mean tumor uptake of 2.1 ± 0.2%ID/g at 15 h post injection. In conclusion, FVIIai was radiolabeled with 64 Cu in single well-defined position of the protein. This method can be utilized to prepare conjugates from serine proteases with the label at a specific position.

Cannabinoid CB2 receptor as a new phototherapy target for the inhibition of tumor growth.

PubMed

Jia, Ningyang; Zhang, Shaojuan; Shao, Pin; Bagia, Christina; Janjic, Jelena M; Ding, Ying; Bai, Mingfeng

2014-06-02

The success of targeted cancer therapy largely relies upon the selection of target and the development of efficient therapeutic agents that specifically bind to the target. In the current study, we chose a cannabinoid CB2 receptor (CB2R) as a new target and used a CB2R-targeted photosensitizer, IR700DX-mbc94, for phototherapy treatment. IR700DX-mbc94 was prepared by conjugating a photosensitizer, IR700DX, to mbc94, whose binding specificity to CB2R has been previously demonstrated. We found that phototherapy treatment using IR700DX-mbc94 greatly inhibited the growth of CB2R positive tumors but not CB2R negative tumors. In addition, phototherapy treatment with nontargeted IR700DX did not show significant therapeutic effect. Similarly, treatment with IR700DX-mbc94 without light irradiation or light irradiation without the photosensitizer showed no tumor-inhibitory effect. Taken together, IR700DX-mbc94 is a promising phototherapy agent with high target-specificity. Moreover, CB2R appears to have great potential as a phototherapeutic target for cancer treatment.

RP-1776, a novel cyclic peptide produced by Streptomyces sp., inhibits the binding of PDGF to the extracellular domain of its receptor.

PubMed

Toki, S; Agatsuma, T; Ochiai, K; Saitoh, Y; Ando, K; Nakanishi, S; Lokker, N A; Giese, N A; Matsuda, Y

2001-05-01

RP-1776, a novel cyclic peptide, was isolated from the culture broth of Streptomyces sp. KY11784. RP-1776 selectively inhibited the binding of PDGF BB to the extracellular domain of the PDGF beta-receptor with an IC50 value of 11 +/- 6 microM. Detailed binding experiments suggested that RP-1776 directly interacts with PDGF BB. RP-1776 inhibited the phosphorylation of the PDGF beta-receptor induced by PDGF BB. These results suggested that RP-1776 antagonizes the signaling of PDGF BB probably through the inhibition of PDGF BB binding to the PDGF beta-receptor.

Two nucleotide binding sites modulate ( sup 3 H) glyburide binding to rat cortex membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, D.E.; Gopalakrishnan, M.; Triggle, D.J.

1991-03-11

The effects of nucleotides on the binding of the ATP-dependent K{sup +}-channel antagonist ({sup 3}H)glyburide (GLB) to rat cortex membranes were examined. Nucleotide triphosphates (NTPs) and nucleotide diphosphate (NDPs) inhibited the binding of GLB. This effect was dependent on the presence of dithiothreitol (DTT). Inhibition of binding by NTPs, with the exception of ATP{gamma}S, was dependent on the presence of Mg{sup 2+}. GLB binding showed a biphasic response to ADP: up to 3 mM, ADP inhibited binding, and above this concentration GLB binding increased rapidly, and was restored to normal levels by 10 mM ADP. In the presence of Mg{supmore » 2+}, ADP did not stimulate binding. Saturation analysis in the presence of Mg{sup 2+} and increasing concentrations of ADP showed that ADP results primarily in a change of the B{sub max} for GLB binding. The differential effects of NTPS and NDPs indicate that two nucleotide binding sites regulate GLB binding.« less

Inhibitors of Fatty Acid Synthase for Prostate Cancer

DTIC Science & Technology

2012-05-01

structure, Figure 3) is highly susceptible to nucelophi lic attack. In addition, well- established Diels - Alder chemistry will b e used to crea te...including each of the precurso rs leading to the compounds. Based on multiple criteria including ability to inhibit recombinant enzyme , ability to...The specificity or hydrophobic channel binds the growing fatty acid chain and guides substrate specificity of the enzyme . The short-chain pock et

Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing.

PubMed

Zhang, Ruowen; Wu, Jiahui; Ferrandon, Sylvain; Glowacki, Katie J; Houghton, Janet A

2016-12-06

The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.

Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain

PubMed Central

Fukunaga, Ryuya; Colpan, Cansu; Han, Bo W; Zamore, Phillip D

2014-01-01

In Drosophila, Dicer-1 produces microRNAs (miRNAs) from pre-miRNAs, whereas Dicer-2 generates small interfering RNAs from long double-stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs. Here, we report that phosphate-dependent substrate discrimination by Dicer-2 reflects dsRNA substrate length. Efficient processing by Dicer-2 of short dsRNA requires a 5′ terminal phosphate and a two-nucleotide, 3′ overhang, but does not require ATP. Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer dsRNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer-2 PAZ domain blocked cleavage of short, but not long, dsRNA. We propose that inorganic phosphate occupies a PAZ domain pocket required to bind the 5′ terminal phosphate of short substrates, blocking their use and restricting pre-miRNA processing in flies to Dicer-1. Our study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme. PMID:24488111

The neuropeptide substance P stimulates the effector functions of platelets.

PubMed Central

Damonneville, M; Monté, D; Auriault, C; Capron, A

1990-01-01

Sensory neuropeptides, such as substance P, appear as potent mediators of various immunological reactions, and inhibit or stimulate a wide range of functions of immune inflammatory cells. Platelets were recently shown to participate as effector cells in an IgE or lymphokine-dependent killing of parasites. Substance P and its carboxy-terminal fragment SP (4-11) induce the cytotoxic activity of platelets towards the larvae of Schistosoma mansoni, respectively, by 90% and 40%, whereas the modified C terminal SP, the SP-free acid, exhibits no effect on the platelets. The neuropeptide effects occur at low doses (10(-8) M), are specific as shown by inhibition studies with a substance P antagonist, the D-SP. Binding data obtained after flow cytofluorometry with FITC-SP lead to the conclusion that SP binds specifically to about 20% of the homogenous population of platelets. Moreover, IgE could modulate the SP-dependent functions of platelets since the pre-incubation with myeloma human IgE or with AP2 monoclonal antibodies--known to inhibit the IgE-dependent killing of these cells-leads to a dramatic decrease of the SP dependent cytotoxic activity of platelets towards the larvae. These findings identify a potent mechanism for nervous system regulation of host defence responses. PMID:1696868

Inhibition of androgen receptor by decoy molecules delays progression to castration-recurrent prostate cancer.

PubMed

Myung, Jae-Kyung; Wang, Gang; Chiu, Helen H L; Wang, Jun; Mawji, Nasrin R; Sadar, Marianne D

2017-01-01

Androgen receptor (AR) is a member of the steroid receptor family and a therapeutic target for all stages of prostate cancer. AR is activated by ligand binding within its C-terminus ligand-binding domain (LBD). Here we show that overexpression of the AR NTD to generate decoy molecules inhibited both the growth and progression of prostate cancer in castrated hosts. Specifically, it was shown that lentivirus delivery of decoys delayed hormonal progression in castrated hosts as indicated by increased doubling time of tumor volume, prolonged time to achieve pre-castrate levels of serum prostate-specific antigen (PSA) and PSA nadir. These clinical parameters are indicative of delayed hormonal progression and improved therapeutic response and prognosis. Decoys reduced the expression of androgen-regulated genes that correlated with reduced in situ interaction of the AR with androgen response elements. Decoys did not reduce levels of AR protein or prevent nuclear localization of the AR. Nor did decoys interact directly with the AR. Thus decoys did not inhibit AR transactivation by a dominant negative mechanism. This work provides evidence that the AR NTD plays an important role in the hormonal progression of prostate cancer and supports the development of AR antagonists that target the AR NTD.

Inhibition of androgen receptor by decoy molecules delays progression to castration-recurrent prostate cancer

PubMed Central

Myung, Jae-Kyung; Wang, Gang; Chiu, Helen H. L.; Wang, Jun; Mawji, Nasrin R.; Sadar, Marianne D.

2017-01-01

Androgen receptor (AR) is a member of the steroid receptor family and a therapeutic target for all stages of prostate cancer. AR is activated by ligand binding within its C-terminus ligand-binding domain (LBD). Here we show that overexpression of the AR NTD to generate decoy molecules inhibited both the growth and progression of prostate cancer in castrated hosts. Specifically, it was shown that lentivirus delivery of decoys delayed hormonal progression in castrated hosts as indicated by increased doubling time of tumor volume, prolonged time to achieve pre-castrate levels of serum prostate-specific antigen (PSA) and PSA nadir. These clinical parameters are indicative of delayed hormonal progression and improved therapeutic response and prognosis. Decoys reduced the expression of androgen-regulated genes that correlated with reduced in situ interaction of the AR with androgen response elements. Decoys did not reduce levels of AR protein or prevent nuclear localization of the AR. Nor did decoys interact directly with the AR. Thus decoys did not inhibit AR transactivation by a dominant negative mechanism. This work provides evidence that the AR NTD plays an important role in the hormonal progression of prostate cancer and supports the development of AR antagonists that target the AR NTD. PMID:28306720

HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids.

PubMed

Cox, Freek; Kwaks, Ted; Brandenburg, Boerries; Koldijk, Martin H; Klaren, Vincent; Smal, Bastiaan; Korse, Hans J W M; Geelen, Eric; Tettero, Lisanne; Zuijdgeest, David; Stoop, Esther J M; Saeland, Eirikur; Vogels, Ronald; Friesen, Robert H E; Koudstaal, Wouter; Goudsmit, Jaap

2016-01-01

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc-FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.

Comparison of functional assays used in the clinical development of a placental malaria vaccine.

PubMed

Pehrson, Caroline; Heno, Kristine K; Adams, Yvonne; Resende, Mafalda; Mathiesen, Line; Soegaard, Max; de Jongh, Willem A; Theander, Thor G; Salanti, Ali; Nielsen, Morten A

2017-01-23

Malaria in pregnancy is associated with significant morbidity in pregnant women and their offspring. Plasmodium falciparum infected erythrocytes (IE) express VAR2CSA that mediates binding to chondroitin sulphate A (CSA) in the placenta. Two VAR2CSA-based vaccines for placental malaria are in clinical development. The purpose of this study was to evaluate the robustness and comparability of binding inhibition assays used in the clinical development of placental malaria vaccines. The ability of sera from animals immunised with different VAR2CSA constructs to inhibit IE binding to CSA was investigated in three in vitro assays using 96-well plates, petri dishes, capillary flow and an ex vivo placental perfusion assay. The inter-assay variation was not uniform between assays and ranged from above ten-fold in the flow assay to two-fold in the perfusion assay. The intra-assay variation was highest in the petri dish assay. A positive correlation between IE binding avidity and the level of binding after antibody inhibition in the petri dish assay indicate that high avidity IE binding is more difficult to inhibit. The highest binding inhibition sensitivity was found in the 96-well and petri dish assays compared to the flow and perfusion assays where binding inhibition required higher antibody titers. The inhibitory capacity of antibodies is not easily translated between assays and the high sensitivity of the 96-well and petri dish assays stresses the need for comparing serial dilutions of serum. Furthermore, IE binding avidity must be in the same range when comparing data from different days. There was an overall concordance in the capacity of antibody-mediated inhibition, when comparing the in vitro assays with the perfusion assay, which more closely represents in vivo conditions. Importantly the ID1-ID2a protein in a liposomal formulation, currently in a phase I trial, effectively induced antibodies that inhibited IE adhesion in placental tissue. Copyright © 2016. Published by Elsevier Ltd.

Selective inhibition of c-Myc/Max dimerization and DNA binding by small molecules.

PubMed

Kiessling, Anke; Sperl, Bianca; Hollis, Angela; Eick, Dirk; Berg, Thorsten

2006-07-01

bZip and bHLHZip protein family members comprise a large fraction of eukaryotic transcription factors and need to bind DNA in order to exert most of their fundamental biological roles. Their binding to DNA requires homo- or heterodimerization via alpha-helical domains, which generally do not contain obvious binding sites for small molecules. We have identified two small molecules, dubbed Mycro1 and Mycro2, which inhibit the protein-protein interactions between the bHLHZip proteins c-Myc and Max. Mycros are the first inhibitors of c-Myc/Max dimerization, which have been demonstrated to inhibit DNA binding of c-Myc with preference over other dimeric transcription factors in vitro. Mycros inhibit c-Myc-dependent proliferation, gene transcription, and oncogenic transformation in the low micromolar concentration range. Our data support the idea that dimeric transcription factors can be druggable even in the absence of obvious small-molecule binding pockets.

Screening and Identification of a Phage Display Derived Peptide That Specifically Binds to the CD44 Protein Region Encoded by Variable Exons.

PubMed

Zhang, Dan; Jia, Huan; Li, Weiming; Hou, Yingchun; Lu, Shaoying; He, Shuixiang

2016-01-01

CD44, especially the isoforms with variable exons (CD44v), is a promising biomarker for the detection of cancer. To develop a CD44v-specific probe, we screened a 7-mer phage peptide library against the CD44v3-v10 protein using an improved subtractive method. The consensus sequences with the highest frequency (designated CV-1) emerged after four rounds of panning. The binding affinity and specificity of the CV-1 phage and the synthesized peptide for the region of CD44 encoded by the variable exons were confirmed using enzyme-linked immunosorbent assay and competitive inhibition assays. Furthermore, the binding of the CV-1 probe to gastric cancer cells and tissues was validated using immunofluorescence and immunohistochemistry assays. CV-1 sensitively and specifically bound to CD44v on cancer cells and tissues. Thus, CV-1 has the potential to serve as a promising probe for cancer molecular imaging and target therapy. © 2015 Society for Laboratory Automation and Screening.

Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.

PubMed

Delafontaine, P; Ku, L; Ververis, J J; Cohen, C; Runge, M S; Alexander, R W

1994-12-01

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.

NFI-Ski interactions mediate transforming growth factor beta modulation of human papillomavirus type 16 early gene expression.

PubMed

Baldwin, Amy; Pirisi, Lucia; Creek, Kim E

2004-04-01

Human papillomaviruses (HPVs) are present in virtually all cervical cancers. An important step in the development of malignant disease, including cervical cancer, involves a loss of sensitivity to transforming growth factor beta (TGF-beta). HPV type 16 (HPV16) early gene expression, including that of the E6 and E7 oncoprotein genes, is under the control of the upstream regulatory region (URR), and E6 and E7 expression in HPV16-immortalized human epithelial cells is inhibited at the transcriptional level by TGF-beta. While the URR contains a myriad of transcription factor binding sites, including seven binding sites for nuclear factor I (NFI), the specific sequences within the URR or the transcription factors responsible for TGF-beta modulation of the URR remain unknown. To identify potential transcription factors and binding sites involved in TGF-beta modulation of the URR, we performed DNase I footprint analysis on the HPV16 URR using nuclear extracts from TGF-beta-sensitive HPV16-immortalized human keratinocytes (HKc/HPV16) treated with and without TGF-beta. Differentially protected regions were found to be located around NFI binding sites. Electrophoretic mobility shift assays, using the NFI binding sites as probes, showed decreased binding upon TGF-beta treatment. This decrease in binding was not due to reduced NFI protein or NFI mRNA levels. Mutational analysis of individual and multiple NFI binding sites in the URR defined their role in TGF-beta sensitivity of the promoter. Overexpression of the NFI family members in HKc/HPV16 decreased the ability of TGF-beta to inhibit the URR. Since the oncoprotein Ski has been shown to interact with and increase the transcriptional activity of NFI and since cellular Ski levels are decreased by TGF-beta treatment, we explored the possibility that Ski may provide a link between TGF-beta signaling and NFI activity. Anti-NFI antibodies coimmunoprecipitated endogenous Ski in nuclear extracts from HKc/HPV16, confirming that NFI and Ski interact in these cells. Ski levels dramatically decreased upon TGF-beta treatment of HKc/HPV16, and overexpression of Ski eliminated the ability of TGF-beta to inhibit the URR. Based on these studies, we propose that TGF-beta inhibition of HPV16 early gene expression is mediated by a decrease in Ski levels, which in turn dramatically reduces NFI activity.

Developing recombinant HPA-1a–specific antibodies with abrogated Fcγ receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia

PubMed Central

Ghevaert, Cedric; Wilcox, David A.; Fang, Juan; Armour, Kathryn L.; Clark, Mike R.; Ouwehand, Willem H.; Williamson, Lorna M.

2008-01-01

Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal generation of antibodies specific for paternal platelet antigens and can lead to fetal intracranial hemorrhage. A SNP in the gene encoding integrin β3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a–specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a–specific scFv (B2) with an IgG1 constant region modified to minimize Fcγ receptor–dependent platelet destruction (G1Δnab). B2G1Δnab saturated HPA-1a+ platelets and substantially inhibited binding of clinical HPA-1a–specific sera to HPA-1a+ platelets. The response of monocytes to B2G1Δnab-sensitized platelets was substantially less than their response to unmodified B2G1, as measured by chemiluminescence. In addition, B2G1Δnab inhibited chemiluminescence induced by B2G1 and HPA-1a–specific sera. In a chimeric mouse model, B2G1 and polyclonal Ig preparations from clinical HPA-1a–specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia. As the Δnab constant region is uninformative in mice, F(ab′)2 B2G1 was used as a proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a–specific antibodies. These results provide rationale for human clinical studies. PMID:18654666

Antagonism of Lidocaine Inhibition by Open-Channel Blockers That Generate Resurgent Na Current

PubMed Central

Bant, Jason S.; Aman, Teresa K.; Raman, Indira M.

2013-01-01

Na channels that generate resurgent current express an intracellular endogenous open-channel blocking protein, whose rapid binding upon depolarization and unbinding upon repolarization minimizes fast and slow inactivation. Na channels also bind exogenous compounds, such as lidocaine, which functionally stabilize inactivation. Like the endogenous blocking protein, these use-dependent inhibitors bind most effectively at depolarized potentials, raising the question of how lidocaine-like compounds affect neurons with resurgent Na current. We therefore recorded lidocaine inhibition of voltage-clamped, tetrodotoxin-sensitive Na currents in mouse Purkinje neurons, which express a native blocking protein, and in mouse hippocampal CA3 pyramidal neurons with and without a peptide from the cytoplasmic tail of NaVβ4 (the β4 peptide), which mimics endogenous open-channel block. To control channel states during drug exposure, lidocaine was applied with rapid-solution exchange techniques during steps to specific voltages. Inhibition of Na currents by lidocaine was diminished by either the β4 peptide or the native blocking protein. In peptide-free CA3 cells, prolonging channel opening with a site-3 toxin, anemone toxin II, reduced lidocaine inhibition; this effect was largely occluded by open-channel blockers, suggesting that lidocaine binding is favored by inactivation but prevented by open-channel block. In constant 100 μM lidocaine, current-clamped Purkinje cells continued to fire spontaneously. Similarly, the β4 peptide reduced lidocaine-dependent suppression of spiking in CA3 neurons in slices. Thus, the open-channel blocking protein responsible for resurgent current acts as a natural antagonist of lidocaine. Neurons with resurgent current may therefore be less susceptible to use-dependent Na channel inhibitors used as local anesthetic, antiarrhythmic, and anticonvulsant drugs. PMID:23486968

Evidence for multiple modes of neutrophil serine protease recognition by the EAP family of Staphylococcal innate immune evasion proteins.

PubMed

Stapels, Daphne A C; Woehl, Jordan L; Milder, Fin J; Tromp, Angelino T; van Batenburg, Aernoud A; de Graaf, Wilco C; Broll, Samuel C; White, Natalie M; Rooijakkers, Suzan H M; Geisbrecht, Brian V

2018-02-01

Neutrophils contain high levels of chymotrypsin-like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so-called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non-covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non-proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes. © 2017 The Protein Society.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Kemin; Johnson, Parker M.; Stols, Lucy

Contact-dependent growth inhibition (CDI) is an important mechanism of intercellular competition between neighboring Gram-negative bacteria. CDI systems encode large surface-exposed CdiA effector proteins that carry a variety of C-terminal toxin domains (CdiA-CTs). All CDI +bacteria also produce CdiI immunity proteins that specifically bind to the cognate CdiA-CT and neutralize its toxin activity to prevent auto-inhibition. Here, the X-ray crystal structure of a CdiI immunity protein fromNeisseria meningitidisMC58 is presented at 1.45 Å resolution. The CdiI protein has structural homology to the Whirly family of RNA-binding proteins, but appears to lack the characteristic nucleic acid-binding motif of this family. Sequence homologymore » suggests that the cognate CdiA-CT is related to the eukaryotic EndoU family of RNA-processing enzymes. A homology model is presented of the CdiA-CT based on the structure of the XendoU nuclease fromXenopus laevis. Molecular-docking simulations predict that the CdiA-CT toxin active site is occluded upon binding to the CdiI immunity protein. Together, these observations suggest that the immunity protein neutralizes toxin activity by preventing access to RNA substrates.« less

GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

PubMed Central

Yasumoto, Hiroaki; Meng, Lingjun; Lin, Tao; Zhu, Qubo; Tsai, Robert Y.L.

2010-01-01

Summary Guanine-nucleotide binding protein 3-like (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1p-null yeasts. To determine the unique function of GNL3L, we identified estrogen receptor-related protein-γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The interaction between GNL3L and ERRγ requires the intermediate domain of GNL3L and the AF2-domain of ERRγ. Gain- and loss-of-function experiments show that GNL3L can inhibit the transcriptional activities of ERR genes in a cell-based reporter system, which does not require the nucleolar localization of GNL3L. We further demonstrate that GNL3L is able to reduce the steroid receptor coactivator (SRC) binding and the SRC-mediated transcriptional coactivation of ERRγ. This work reveals a novel mechanism that negatively regulates the transcriptional function of ERRγ by GNL3L through coactivator competition. PMID:17623774

Rational design and synthesis of 2-anilinopyridinyl-benzothiazole Schiff bases as antimitotic agents.

PubMed

Shaik, Thokhir B; Hussaini, S M Ali; Nayak, V Lakshma; Sucharitha, M Lakshmi; Malik, M Shaheer; Kamal, Ahmed

2017-06-01

Based on our previous results and literature precedence, a series of 2-anilinopyridinyl-benzothiazole Schiff bases were rationally designed by performing molecular modeling experiments on some selected molecules. The binding energies of the docked molecules were better than the E7010, and the Schiff base with trimethoxy group on benzothiazole moiety, 4y was the best. This was followed by the synthesis of a series of the designed molecules by a convenient synthetic route and evaluation of their anticancer potential. Most of the compounds have shown significant growth inhibition against the tested cell lines and the compound 4y exhibited good antiproliferative activity with a GI 50 value of 3.8µM specifically against the cell line DU145. In agreement with the docking results, 4y exerted cytotoxicity by the disruption of the microtubule dynamics by inhibiting tubulin polymerization via effective binding into colchicine domain, comparable to E7010. Detailed binding modes of 4y with colchicine binding site of tubulin were studied by molecular docking. Furthermore, 4y induced apoptosis as evidenced by biological studies like mitochondrial membrane potential, caspase-3, and Annexin V-FITC assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

NASA Astrophysics Data System (ADS)

Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

2014-12-01

The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

Ropizine concurrently enhances and inhibits ( sup 3 H) dextromethorpan binding to different structures of the guinea pig brain: Autoradiographic evidence for multiple binding sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Canoll, P.D.; Smith, P.R.; and Musacchio, J.M.

1990-01-01

Ropizine produces a simultaneous enhancement and inhibition of ({sup 3}H) dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity ({sup 3}H)DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhances ({sup 3}H)DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+){minus} pentazocine, has not been fullymore » characterized. This study demonstrates that the biphasic effects to ropizine are due, at least in part, to the effects of ropizine on two different types of ({sup 3}H)DM binding sites. However, this study does not rule out that the common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.« less

Action of tremorgenic mycotoxins on GABA/sub A/ receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gant, D.B.; Cole, R.J.; Valdes, J.J.

1987-11-09

The effects of four tremorgenic and one nontremorgenic mycotoxins were studied on ..gamma..-aminobutyric acid (GABA/sub A/) receptor binding and function in rat brain and on binding of a voltage-operated Cl/sup -/ channel in Torpedo electric organ. None of the mycotoxins had significant effect on (/sup 3/H)muscimol or (/sup 3/H)flunitrazepam binding to the GAMA/sup A/ receptor. However, only the four tremorgenic mycotoxins inhibited GABA-induced /sup 36/Cl/sup -/ influx and (/sup 35/S)t-butylbicyclophosphorothionate ((/sup 35/S)TBPS) binding in rate brain membranes, while the nontremorgenic verruculotoxin had no effect. Inhibition of (/sup 35/S)TBPS binding by paspalinine was non-competitive. This suggests that tremorgenic mycotoxins inhibit GABA/submore » A/ receptor function by binding close to the receptor's Cl/sup -/ channel. On the voltage-operated Cl/sup -/ channel, only high concentrations of verruculogen and verruculotoxin caused significant inhibition of the channel's binding of (/sup 35/S)TBPS. The data suggest that the tremorgenic action of these mycotoxins may be due in part to their inhibition of GABA/sub A/ receptor function. 21 references, 4 figures, 2 tables.« less

Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.

PubMed

Chang, C P; Hüsler, T; Zhao, J; Wiedmer, T; Sims, P J

1994-10-21

The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

Effects of Lectins on initial attachment of cariogenic Streptococcus mutans.

PubMed

Ito, Takashi; Yoshida, Yasuhiro; Shiota, Yasuyoshi; Ito, Yuki; Yamamoto, Tadashi; Takashiba, Shogo

2018-02-01

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.