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Sample records for binding protein 1c

  1. Protein kinase Cbeta mediates hepatic induction of sterol-regulatory element binding protein-1c by insulin

    OpenAIRE

    Yamamoto, Takashi; Watanabe, Kazuhisa; Inoue, Noriyuki; Nakagawa, Yoshimi; Ishigaki, Naomi; Matsuzaka, Takashi; Takeuchi, Yoshinori; Kobayashi, Kazuto; Yatoh, Shigeru; Takahashi, Akimitsu; Suzuki, Hiroaki; Yahagi, Naoya; Gotoda, Takanari; Yamada, Nobuhiro; Shimano, Hitoshi

    2010-01-01

    Sterol-regulatory element binding protein-1c (SREBP-1c) is a transcription factor that controls lipogenesis in the liver. Hepatic SREBP-1c is nutritionally regulated, and its sustained activation causes hepatic steatosis and insulin resistance. Although regulation of SREBP-1c is known to occur at the transcriptional level, the precise mechanism by which insulin signaling activates SREBP-1c promoter remains to be elucidated. Here we show that protein kinase C beta (PKCbeta) is a key mediator o...

  2. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation.

    Science.gov (United States)

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N; Bridges, Dave; Park, Edwards A; Cole, Robert N; Elam, Marshall B; Raghow, Rajendra

    2016-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c-SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCF(Fbw7) ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  3. In vivo promoter analysis on refeeding response of hepatic sterol regulatory element-binding protein-1c expression

    International Nuclear Information System (INIS)

    Sterol regulatory element-binding protein (SREBP)-1c is the master regulator of lipogenic gene expression in liver. The mRNA abundance of SREBP-1c is markedly induced when animals are refed after starvation, although the regulatory mechanism is so far unknown. To investigate the mechanism of refeeding response of SREBP-1c gene expression in vivo, we generated a transgenic mouse model that carries 2.2 kb promoter region fused to the luciferase reporter gene. These transgenic mice exhibited refeeding responses of the reporter in liver and adipose tissues with extents essentially identical to those of endogenous SREBP-1c mRNA. The same results were obtained from experiments using adenovirus-mediated SREBP-1c-promoter-luciferase fusion gene transduction to liver. These data demonstrate that the regulation of SREBP-1c gene expression is at the transcription level, and that the 2.2 kb 5'-flanking region is sufficient for this regulation. Moreover, when these transgenic or adenovirus-infected mice were placed on insulin-depleted state by streptozotocin treatment, the reporter expression was upregulated as strongly as in control mice, demonstrating that this regulation is not dominated by serum insulin level. These mice are the first models to provide the mechanistic insight into the transcriptional regulation of SREBP-1c gene in vivo

  4. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    International Nuclear Information System (INIS)

    Research highlights: → A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. → HCV-3a NS5A increases mature SREBP-1c protein level. → HCV-3a NS5A activates SREBP-1c transcription. → Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. → Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  5. Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells.

    OpenAIRE

    Shimomura, I; Shimano, H; Horton, J D; Goldstein, J L; Brown, M S

    1997-01-01

    The 5' end of the mRNA-encoding sterol regulatory element binding protein-1 (SREBP-1) exists in two forms, designated 1a and 1c. The divergence results from the use of two transcription start sites that produce two separate 5' exons, each of which is spliced to a common exon 2. Here we show that the ratio of SREBP-1c to 1a transcripts varies markedly among organs of the adult mouse. At one extreme is the liver, in which the 1c transcript predominates by a 9:1 ratio. High 1c:1a ratios are also...

  6. Honokiol reverses alcoholic fatty liver by inhibiting the maturation of sterol regulatory element binding protein-1c and the expression of its downstream lipogenesis genes

    International Nuclear Information System (INIS)

    Ethanol induces hepatic steatosis via a complex mechanism that is not well understood. Among the variety of molecules that have been proposed to participate in this mechanism, the sterol regulatory element (SRE)-binding proteins (SREBPs) have been identified as attractive targets for therapeutic intervention. In the present study, we evaluated the effects of honokiol on alcoholic steatosis and investigated its possible effect on the inhibition of SREBP-1c maturation. In in vitro studies, H4IIEC3 rat hepatoma cells developed increased lipid droplets when exposed to ethanol, but co-treatment with honokiol reversed this effect. Honokiol inhibited the maturation of SREBP-1c and its translocation to the nucleus, the binding of nSREBP-1c to SRE or SRE-related sequences of its lipogenic target genes, and the expression of genes for fatty acid synthesis. In contrast, magnolol, a structural isomer of honokiol, had no effect on nSREBP-1c levels. Male Wistar rats fed with a standard Lieber-DeCarli ethanol diet for 4 weeks exhibited increased hepatic triglyceride and decreased hepatic glutathione levels, with concomitantly increased serum alanine aminotransferase and TNF-α levels. Daily administration of honokiol (10 mg/kg body weight) by gavage during the final 2 weeks of ethanol treatment completely reversed these effects on hepatotoxicity markers, including hepatic triglyceride, hepatic glutathione, and serum TNF-α, with efficacious abrogation of fat accumulation in the liver. Inhibition of SREBP-1c protein maturation and of the expression of Srebf1c and its target genes for hepatic lipogenesis were also observed in vivo. A chromatin immunoprecipitation assay demonstrated inhibition of specific binding of SREBP-1c to the Fas promoter by honokiol in vivo. These results demonstrate that honokiol has the potential to ameliorate alcoholic steatosis by blocking fatty acid synthesis regulated by SREBP-1c

  7. Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

    Directory of Open Access Journals (Sweden)

    Cyr Janet L

    2002-11-01

    Full Text Available Abstract Background Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3 in its neck region; we identified a fourth IQ domain (IQ4, located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. Results In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much weaker. Ca2+ substantially weakened the calmodulin-peptide affinity for all of the IQ peptides except IQ3. To reveal how calmodulin bound to the linearly arranged IQ domains of the myosin-1c neck, we used hydrodynamic measurements to determine the stoichiometry of complexes of calmodulin and myosin-1c. Purified myosin-1c and T701-Myo1c (a myosin-1c fragment with all four IQ domains and the C-terminal tail each bound 2–3 calmodulin molecules. At a physiologically relevant temperature (25°C and under low-Ca2+ conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 μM free calmodulin. Ca2+ dissociated nearly all calmodulins from T701-Myo1c at 25°C; one calmodulin was retained if 5 μM free calmodulin was present. Conclusions We inferred from these data that at 25°C and normal cellular concentrations of calmodulin, calmodulin is bound to IQ1, IQ2, and IQ3 of myosin-1c when Ca2+ is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca2+ elevation, all calmodulin except that bound to IQ3 should dissociate.

  8. Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

    OpenAIRE

    Cyr Janet L; Gillespie Peter G

    2002-01-01

    Abstract Background Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3) in its neck region; we identified a fourth IQ domain (IQ4), located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. Results In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much...

  9. Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

    OpenAIRE

    Ford, A M; Bennett, C.A; Healy, L E; TOWATARI, M.; Greaves, M F; Enver, T

    1996-01-01

    We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated ...

  10. Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport.

    Science.gov (United States)

    Rehan, Medhat; Furnholm, Teal; Finethy, Ryan H; Chu, Feixia; El-Fadly, Gomaah; Tisa, Louis S

    2014-09-01

    Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller "leaf-like" structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics' analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu(2+) stress. After 5 days of Cu(2+) stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu(2+)-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu(2+)-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins. PMID:24903815

  11. The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation.

    Science.gov (United States)

    Chen, Yu-Tsung Shane; Wu, Jianhong; Modrich, Paul; Hsieh, Tao-Shih

    2016-06-17

    Eukaryotic topoisomerase 2 (Top2) and one of its interacting partners, topoisomerase IIβ binding protein 1 (TopBP1) are two proteins performing essential cellular functions. We mapped the interacting domains of these two proteins using co-immunoprecipitation and pulldown experiments with truncated or mutant Drosophila Top2 with various Ser-to-Ala substitutions. We discovered that the last 20 amino acids of Top2 represent the key region for binding with Mus101 (the Drosophila homolog of TopBP1) and that phosphorylation of Ser-1428 and Ser-1443 is important for Top2 to interact with the N terminus of Mus101, which contains the BRCT1/2 domains. The interaction between Mus101 and the Top2 C-terminal regulatory domain is phosphorylation-dependent because treatment with phosphatase abolishes their association in pulldown assays. The binding affinity of N-terminal Mus101 with a synthetic phosphorylated peptide spanning the last 25 amino acids of Top2 (with Ser(P)-1428 and Ser(P)-1443) was determined by surface plasmon resonance with a Kd of 0.57 μm In an in vitro decatenation assay, Mus101 can specifically reduce the decatenation activity of Top2, and dephosphorylation of Top2 attenuates this response. Next, we endeavored to establish a cellular system for testing the biological function of Top2-Mus101 interaction. Top2-silenced S2 cells rescued by Top2Δ20, Top2 with 20 amino acids truncated from the C terminus, developed abnormally high chromosome numbers, which implies that Top2-Mus101 interaction is important for maintaining the fidelity of chromosome segregation during mitosis. PMID:27129233

  12. Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin

    DEFF Research Database (Denmark)

    Hansen, G. A. W.; Ludvigsen, M.; Jacobsen, C.;

    2015-01-01

    Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify...... interacted with both proteins with an estimated dissociation constant at 1 mu M for reticulocalbin and 150 nM for calumenin. The interaction, at least for calumenin, was dependent upon the presence of Ca2+ with strong interaction at 3.5 mM while no detectable interaction could be found at 0.1 mM. Grp75 binds...

  13. BINDING ISOTHERMS SURFACTANT-PROTEINS

    OpenAIRE

    Elena Irina Moater; Cristiana Radulescu; Ionica Ionita

    2011-01-01

    The interactions between surfactants and proteins shows some similarities with interactions between surfactants and polymers, but the hydrophobic amphoteric nature of proteins and their secondary and tertiary structure components make them different from conventional polymer systems. Many studies from the past about surfactant - proteins bonding used the dialysis techniques. Other techniques used to determine the binding isotherm, included ultrafiltration, ultracentrifugation, potentiometry, ...

  14. Using poly(3-aminophenylboronic acid) thin film with binding-induced ion flux blocking for amperometric detection of hemoglobin A1c.

    Science.gov (United States)

    Wang, Jen-Yuan; Chou, Tse-Chuan; Chen, Lin-Chi; Ho, Kuo-Chuan

    2015-01-15

    This study reports a novel enzyme-free, label-free amperometric method for direct detection of hemoglobin A1c (Hb(A1c)), a potent biomarker for diabetes diagnosis and prognosis. The method relies on an electrode modified with poly(3-aminophenylboronic acid) (PAPBA) nanoparticles (20-50 nm) and a sensing scheme named "binding-induced ion flux blocking." The PAPBA nanoparticles were characterized by FT-IR, XPS, TEM, and SEM. Being a polyaniline derivative, PAPBA showed an ion-dependent redox behavior, in which insertion or extraction of ions into or out of PABPA occurred for charge balance during the electron transfer process. The polymer allowed Hb(A1c) selectively bound to its surface via forming the cis-diol linkage between the boronic acid and sugar moieties. Voltammetric analyses showed that Hb(A1c) binding decreased the redox current of PAPBA; however, the binding did not alter the redox potentials and the apparent diffusivities of ions. This suggests that the redox current of PAPBA decreased due to an Hb(A1c) binding-induced ion flux blocking mechanism, which was then verified and characterized through an in situ electrochemical quartz crystal microbalance (EQCM) study. Assay with Hb(A1c) by differential pulse voltammetry (DPV) indicates that the peak current of a PAPBA electrode has a linear dependence on the logarithm of Hb(A1c) concentration ranging from 0.975 to 156 μM. The Hb(A1c) assay also showed high selectivity against ascorbic acid, dopamine, uric acid, glucose and bovine serum albumin. This study has demonstrated a new method for developing an electrochemical Hb(A1c) biosensor and can be extended to other label-free, indicator-free protein biosensors based on a similar redox polymer electrode. PMID:25113050

  15. Protein Dynamics in an RNA Binding Protein

    Science.gov (United States)

    Hall, Kathleen

    2006-03-01

    Using ^15N NMR relaxation measurements, analyzed with the Lipari-Szabo formalism, we have found that the human U1A RNA binding protein has ps-ns motions in those loops that make contact with RNA. Specific mutations can alter the extent and pattern of motions, and those proteins inevitably lose RNA binding affinity. Proteins with enhanced mobility of loops and termini presumably lose affinity due to increased conformational sampling by those parts of the protein that interact directly with RNA. There is an entropic penalty associated with locking down those elements upon RNA binding, in addition to a loss of binding efficiency caused by the increased number of conformations adopted by the protein. However, in addition to local conformational heterogeneity, analysis of molecular dynamics trajectories by Reorientational Eigenmode Dynamics reveals that loops of the wild type protein undergo correlated motions that link distal sites across the binding surface. Mutations that disrupt correlated motions result in weaker RNA binding, implying that there is a network of interactions across the surface of the protein. (KBH was a Postdoctoral Fellow with Al Redfield from 1985-1990). This work was supported by the NIH (to KBH) and NSF (SAS).

  16. Grafting of protein-protein binding sites

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Ca and Cb atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

  17. Photoconversion and fluorescence properties of a red/green-type cyanobacteriochrome AM1_C0023g2 that binds not only phycocyanobilin but also biliverdin.

    Directory of Open Access Journals (Sweden)

    Keiji Fushimi

    2016-04-01

    Full Text Available Cyanobacteriochromes (CBCRs are distantly related to the red/far-red responsive phytochromes. Red/green-type CBCRs are widely distributed among various cyanobacteria. The red/green-type CBCRs covalently bind phycocyanobilin (PCB and show red/green reversible photoconversion. Recent studies revealed that some red/green-type CBCRs from chlorophyll d-bearing cyanobacterium Acaryochloris marina covalently bind not only PCB but also biliverdin (BV. The BV-binding CBCRs show far-red/orange reversible photoconversion. Here, we identified another CBCR (AM1_C0023g2 from A. marina that also covalently binds not only PCB but also BV with high binding efficiencies, although BV chromophore is unstable in the presence of urea. Replacement of Ser334 with Gly resulted in significant improvement in the yield of the BV-binding holoprotein, thereby ensuring that the mutant protein is a fine platform for future development of optogenetic switches. We also succeeded in detecting near-infrared fluorescence from mammalian cells harboring PCB-binding AM1_C0023g2 whose fluorescence quantum yield is 3.0%. Here the PCB-binding holoprotein is shown as a platform for future development of fluorescent probes.

  18. Photoconversion and Fluorescence Properties of a Red/Green-Type Cyanobacteriochrome AM1_C0023g2 That Binds Not Only Phycocyanobilin But Also Biliverdin.

    Science.gov (United States)

    Fushimi, Keiji; Nakajima, Takahiro; Aono, Yuki; Yamamoto, Tatsuro; Ni-Ni-Win; Ikeuchi, Masahiko; Sato, Moritoshi; Narikawa, Rei

    2016-01-01

    Cyanobacteriochromes (CBCRs) are distantly related to the red/far-red responsive phytochromes. Red/green-type CBCRs are widely distributed among various cyanobacteria. The red/green-type CBCRs covalently bind phycocyanobilin (PCB) and show red/green reversible photoconversion. Recent studies revealed that some red/green-type CBCRs from chlorophyll d-bearing cyanobacterium Acaryochloris marina covalently bind not only PCB but also biliverdin (BV). The BV-binding CBCRs show far-red/orange reversible photoconversion. Here, we identified another CBCR (AM1_C0023g2) from A. marina that also covalently binds not only PCB but also BV with high binding efficiencies, although BV chromophore is unstable in the presence of urea. Replacement of Ser334 with Gly resulted in significant improvement in the yield of the BV-binding holoprotein, thereby ensuring that the mutant protein is a fine platform for future development of optogenetic switches. We also succeeded in detecting near-infrared fluorescence from mammalian cells harboring PCB-binding AM1_C0023g2 whose fluorescence quantum yield is 3.0%. Here the PCB-binding holoprotein is shown as a platform for future development of fluorescent probes. PMID:27242674

  19. Soy isoflavones increase quinone reductase in hepa-1c1c7 cells via estrogen receptor beta and nuclear factor erythroid 2-related factor 2 binding to the antioxidant response element.

    Science.gov (United States)

    Froyen, Erik B; Steinberg, Francene M

    2011-09-01

    Soy protein and isoflavones (genistein and daidzein) have been demonstrated to increase quinone reductase (QR) activity, protein, and mRNA in animal and cell culture models. However, their mechanism of action has not been completely characterized. Additionally, it has not been determined if equol, a daidzein metabolite, can modulate QR activity and expression. Estrogen receptor beta (ERβ) is thought to be involved in stimulating QR gene transcription by anti-estrogens and phytoestrogens, along with nuclear factor erythroid 2-related factor 2 (Nrf2). This study tested the hypothesis that genistein, daidzein and equol increase quinone reductase activity, protein and mRNA via ERβ and Nrf2 binding to the QR antioxidant response element (ARE). QR expression and activity were determined using TaqMan polymerase chain reaction, protein immunoblots and activity assays. Molecular events were investigated using luciferase reporter gene assays and chromatin immunoprecipitation (ChIP). Hepa-1c1c7 cells were treated with control [0.1% (v:v) dimethyl sulfoxide (DMSO)]; 1 μmol/L β-naphthoflavone (positive control); 5 μmol/L resveratrol (ChIP positive control for ERβ binding) and 1, 5 and 25 μmol/L genistein, daidzein or equol. Treatment durations were 1 h (ChIP), 24 h (mRNA and luciferase assays) and 24 and 48 h (protein and activity). Genistein, daidzein and equol increased QR activity, protein and mRNA, with daidzein and equol having more of an impact at physiologic concentrations (1 and 5 μmol/L) compared to genistein. Furthermore, the study results demonstrate that genistein, daidzein and equol interact with the QR ARE and that daidzein and equol act via both ERβ and Nrf2 binding strongly to the QR ARE. PMID:21167702

  20. Hepatic expression of sterol regulatory element binding protein 1c and its significance in patients with non-alcoholic fatty liver disease%非酒精性脂肪肝患者肝脏固醇调节元件结合蛋白1c的表达及意义

    Institute of Scientific and Technical Information of China (English)

    孙振江; 石伟珍; 王宇芳; 施军平; 过建春

    2014-01-01

    Objective To investigate the hepatic expression of sterol regulatory element binding protein (SREBP)-1c in patients with non-alcoholic fatty liver disease (NAFLD) and its significance.Methods Twenty patients with NAFLD were included,and 20 age/sex matched asymptomatic HBV carriers (ASC) without steatosis evidenced by liver biopsy were used as control in the same time scale.Hepatic SREBP1c was detected using SP method of immunohistochemistry.An automatic biochemical analyzer (Hitachi 7060) was used to measure serum levels of lipids (TG,CHO),fasting blood glucose (FBG) and liver enzymes (AST,A LT,and GGT),and electrochemiluminescence was used to determine fasting insulin (FINS).Results Compared with the control group,NAFLD patients showed diffusely hepatic steatosis and a significantly higher expression of hepatic SREBP1c (P < 0.05).Consistently,serum levels of ALT,AST,TG,CHO,FSG and FINS all increased than those of control group (P < 0.05).Conclusions Hepatic expression of SREBP1c increased significantly in patients with NAFLD,and this over expression may be involve in fatty degeneration of the livers in human.%目的 观察非酒精性脂肪性肝病(NAFLD)患者肝组织固醇调节元件结合蛋白(SREBP1c)的表达变化,探讨其在NAFLD病理变化形成中的作用.方法 研究对象为NAFLD患者20例,20例年龄性别匹配的同期住院行肝活检无活动肝病组织学证据且肝脂肪变<5%的慢性HBV携带者(ASC)设为对照组.免疫组织化学染色检测两组患者肝组织SREBP1c的表达,并比较血脂(TG、CHO)、空腹血糖(FBG)、空腹胰岛素(FINS)和血清肝脏酶谱(AST、ALT、GGT)等的组间差异.结果 与对照组患者比较,NAFLD组肝组织呈不同程度的弥漫性肝细胞脂肪变性,肝组织SREBP1c表达明显升高(P<0.05),与此一致,血清ALT、AST、TG、CHO、FBG、FINS水平升高(P均<0.05).结论 NAFLD患者肝组织SREBP1 c表达增高,在人NAFLD脂肪变形成过程中起重要作用.

  1. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen; Gammeltoft, Steen

    2012-01-01

    Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

  2. Erythropoietin binding protein from mammalian serum

    Energy Technology Data Exchange (ETDEWEB)

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  3. Erythropoietin binding protein from mammalian serum

    Energy Technology Data Exchange (ETDEWEB)

    Clemons, Gisela K. (Berkeley, CA)

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  4. Overexpression of AtBMI1C, a polycomb group protein gene, accelerates flowering in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available Polycomb group protein (PcG-mediated gene silencing is emerging as an essential developmental regulatory mechanism in eukaryotic organisms. PcGs inactivate or maintain the silenced state of their target chromatin by forming complexes, including Polycomb Repressive Complex 1 (PRC1 and 2 (PRC2. Three PRC2 complexes have been identified and characterized in Arabidopsis; of these, the EMF and VRN complexes suppress flowering by catalyzing the trimethylation of lysine 27 on histone H3 of FLOWER LOCUS T (FT and FLOWER LOCUS C (FLC. However, little is known about the role of PRC1 in regulating the floral transition, although AtRING1A, AtRING1B, AtBMI1A, and AtBMI1B are believed to regulate shoot apical meristem and embryonic development as components of PRC1. Moreover, among the five RING finger PcGs in the Arabidopsis genome, four have been characterized. Here, we report that the fifth, AtBMI1C, is a novel, ubiquitously expressed nuclear PcG protein and part of PRC1, which is evolutionarily conserved with Psc and BMI1. Overexpression of AtBMI1C caused increased H2A monoubiquitination and flowering defects in Arabidopsis. Both the suppression of FLC and activation of FT were observed in AtBMI1C-overexpressing lines, resulting in early flowering. No change in the H3K27me3 level in FLC chromatin was detected in an AtBMI1C-overexpressing line. Our results suggest that AtBMI1C participates in flowering time control by regulating the expression of FLC; moreover, the repression of FLC by AtBMI1C is not due to the activity of PRC2. Instead, it is likely the result of PRC1 activity, into which AtBMI1C is integrated.

  5. S14 protein in breast cancer cells: Direct evidence of regulation by SREBP-1c, superinduction with progestin, and effects on cell growth

    International Nuclear Information System (INIS)

    Most breast cancers exhibit brisk lipogenesis, and require it for growth. S14 is a lipogenesis-related nuclear protein that is overexpressed in most breast cancers. Sterol response element-binding protein-1c (SREBP-1c) is required for induction of lipogenesis-related genes, including S14 and fatty acid synthase (FAS), in hepatocytes, and correlation of SREBP-1c and FAS expression suggested that SREBP-1c drives lipogenesis in tumors as well. We directly tested the hypothesis that SREBP-1c drives S14 expression and mediates lipogenic effects of progestin in T47D breast cancer cells. Dominant-negative SREBP-1c inhibited induction of S14 and FAS mRNAs by progestin, while active SREBP-1c induced without hormone and superinduced in its presence. Changes in S14 mRNA were reflected in protein levels. A lag time and lack of progestin response elements indicated that S14 and FAS gene activation by progestin is indirect. Knockdown of S14 reduced, whereas overexpression stimulated, T47D cell growth, while nonlipogenic MCF10a mammary epithelial cells were not growth-inhibited. These data directly demonstrate that SREBP-1c drives S14 gene expression in breast cancer cells, and progestin magnifies that effect via an indirect mechanism. This supports the prediction, based on S14 gene amplification and overexpression in breast tumors, that S14 augments breast cancer cell growth and survival

  6. Characterization of sensory neuron subpopulations selectively expressing green fluorescent protein in phosphodiesterase 1C BAC transgenic mice

    Directory of Open Access Journals (Sweden)

    Anderson Rebecca L

    2006-05-01

    Full Text Available Abstract Background The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c BAC transgenic mice in which a green fluorescent protein (GFP reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord. Results Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative. A small proportion of both non-peptidergic (IB4-binding and peptidergic (CGRP immunoreactive subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X3. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn. Conclusion The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.

  7. Advances on Plant Pathogenic Mycotoxin Binding Proteins

    Institute of Scientific and Technical Information of China (English)

    WANG Chao-hua; DONG Jin-gao

    2002-01-01

    Toxin-binding protein is one of the key subjects in plant pathogenic mycotoxin research. In this paper, new advances in toxin-binding proteins of 10 kinds of plant pathogenic mycotoxins belonging to Helminthosporium ,Alternaria ,Fusicoccum ,Verticillium were reviewed, especially the techniques and methods of toxin-binding proteins of HS-toxin, HV-toxin, HMT-toxin, HC-toxin. It was proposed that the isotope-labeling technique and immunological chemistry technique should be combined together in research of toxin-binding protein, which will be significant to study the molecular recognition mechanism between host and pathogenic fungus.

  8. Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1.

    Science.gov (United States)

    Povelones, Michael; Upton, Leanna M; Sala, Katarzyna A; Christophides, George K

    2011-04-01

    Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition. PMID:21533217

  9. Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1.

    Directory of Open Access Journals (Sweden)

    Michael Povelones

    2011-04-01

    Full Text Available Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition.

  10. A heterodimeric complex of the LRR proteins LRIM1 and APL1C regulates complement-like immunity in Anopheles gambiae

    Energy Technology Data Exchange (ETDEWEB)

    Baxter, Richard H.G.; Steinert, Stefanie; Chelliah, Yogarany; Volohonsky, Gloria; Levashina, Elena A.; Deisenhofer, Johann (CNRS-UMR); (UTSMC)

    2012-01-20

    The leucine-rich repeat (LRR) proteins LRIM1 and APL1C control the function of the complement-like protein TEP1 in Anopheles mosquitoes. The molecular structure of LRIM1 and APL1C and the basis of their interaction with TEP1 represent a new type of innate immune complex. The LRIM1/APL1C complex specifically binds and solubilizes a cleaved form of TEP1 without an intact thioester bond. The LRIM1 and APL1C LRR domains have a large radius of curvature, glycosylated concave face, and a novel C-terminal capping motif. The LRIM1/APL1C complex is a heterodimer with a single intermolecular disulfide bond. The structure of the LRIM1/APL1C heterodimer reveals an interface between the two LRR domains and an extensive C-terminal coiled-coil domain. We propose that a cleaved form of TEP1 may act as a convertase for activation of other TEP1 molecules and that the LRIM1/APL1C heterodimer regulates formation of this TEP1 convertase.

  11. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  12. Penicillin-Binding Protein Imaging Probes

    OpenAIRE

    Kocaoglu, Ozden; Carlson, Erin E.

    2013-01-01

    Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, the apparent functional redundancy of the ~5–15 proteins that most bacteria possess makes determination of their individual roles difficult. Existing techniques to st...

  13. Calmodulin Binding Proteins and Alzheimer's Disease.

    Science.gov (United States)

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A

    2015-01-01

    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  14. Molecular cloning and expression of the carbohydrate response element binding protein gene and related genes involved in hepatic lipogenesis during post-hatch development of broiler chickens

    Science.gov (United States)

    Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Responding to changes in the level of glucose (ChREBP) and insulin (SREBP-1c), these two transcripti...

  15. Structure of the DNA-bound BRCA1 C-terminal Region from Human Replication Factor C p140 and Model of the Protein-DNA Complex

    OpenAIRE

    2010-01-01

    BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5′-phosphoryl...

  16. Mercury-binding proteins of Mytilus edulis

    Energy Technology Data Exchange (ETDEWEB)

    Roesijadi, G.; Morris, J. E.; Calabrese, A.

    1981-11-01

    Mytilus edulis possesses low molecular weight, mercury-binding proteins. The predominant protein isolated from gill tissue is enriched in cysteinyl residues (8%) and possesses an amino acid composition similar to cadmium-binding proteins of mussels and oysters. Continuous exposure of mussels to 5 ..mu..g/l mercury results in spillover of mercury from these proteins to high molecular weight proteins. Antibodies to these proteins have been isolated, and development of immunoassays is presently underway. Preliminary studies to determine whether exposure of adult mussels to mercury will result in induction of mercury-binding proteins in offspring suggest that such proteins occur in larvae although additional studies are indicated for a conclusive demonstration.

  17. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  18. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  19. Alcohol Binding to the Odorant Binding Protein LUSH: Multiple Factors Affecting Binding Affinities

    OpenAIRE

    Ader, Lauren; Jones, David N. M.; Lin, Hai

    2010-01-01

    Density function theory (DFT) calculations have been carried out to investigate the binding of alcohols to the odorant binding protein LUSH from Drosophila melanogaster. LUSH is one of the few proteins known to bind to ethanol at physiologically relevant concentrations and where high-resolution structural information is available for the protein bound to alcohol at these concentrations. The structures of the LUSH–alcohol complexes identify a set of specific hydrogen-bonding interactions as cr...

  20. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns.

    OpenAIRE

    Swanson, M S; Dreyfuss, G

    1988-01-01

    The binding of hnRNP proteins to pre-mRNAs in nuclear extracts, and as isolated proteins, was studied by using monoclonal antibody immunopurification of hnRNP proteins bound to RNase T1-generated fragments. Several major hnRNP proteins, A1, C and D, bind specifically to the 3' end of introns within a region containing the conserved polypyrimidine stretch between the branch site and the 3' splice site. Mutations which alter the conserved 3' splice site dinucleotide AG strongly impair or abolis...

  1. Calcineurin homologous protein: a multifunctional Ca2+-binding protein family

    OpenAIRE

    Di Sole, Francesca; Vadnagara, Komal; MOE, ORSON W.; Babich, Victor

    2012-01-01

    The calcineurin homologous protein (CHP) belongs to an evolutionarily conserved Ca2+-binding protein subfamily. The CHP subfamily is composed of CHP1, CHP2, and CHP3, which in vertebrates share significant homology at the protein level with each other and between other Ca2+-binding proteins. The CHP structure consists of two globular domains containing from one to four EF-hand structural motifs (calcium-binding regions composed of two helixes, E and F, joined by a loop), the myristoylation, a...

  2. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan;

    2005-01-01

    to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is...

  3. Affinity purification of proteins binding to GST fusion proteins.

    Science.gov (United States)

    Swaffield, J C; Johnston, S A

    2001-05-01

    This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding. PMID:18265191

  4. L-1-C-11-tyrosine PET in patients with laryngeal carcinomas : Comparison of standardized uptake value and protein synthesis rate

    NARCIS (Netherlands)

    de Boer, [No Value; Pruim, J; van der Laan, BFAM; Que, TH; Willemsen, ATM; Albers, FWJ; Vaalburg, W

    2003-01-01

    PET with L-1-C-11-tyrosine (TYR) can measure and quantify increased protein synthesis in tumor tissue in vivo. For quantification of the protein synthesis rate (PSR), arterial cannulation with repeated blood sampling to obtain the plasma input function and a dynamic TYR PET study to calculate a time

  5. Treponema pallidum Fibronectin-Binding Proteins

    OpenAIRE

    Cameron, Caroline E.; Brown, Elizabeth L.; Kuroiwa, Janelle M. Y.; Schnapp, Lynn M.; Brouwer, Nathan L.

    2004-01-01

    Putative adhesins were predicted by computer analysis of the Treponema pallidum genome. Two treponemal proteins, Tp0155 and Tp0483, demonstrated specific attachment to fibronectin, blocked bacterial adherence to fibronectin-coated slides, and supported attachment of fibronectin-producing mammalian cells. These results suggest Tp0155 and Tp0483 are fibronectin-binding proteins mediating T. pallidum-host interactions.

  6. 牙鲆胰岛素样生长因子结合蛋白IGFBP-1cDNA全长的克隆及表达分析%The full-length cDNA cloning and gene expression analysis of insulin-like growth factor binding protein 1 (IGFBP-1)in Japanese flounder ( Paralichthys olivaceus )

    Institute of Scientific and Technical Information of China (English)

    翟万营; 张俊玲; 施志仪; 李巍

    2012-01-01

    The full-length cDNA of insulin- like growth factor binding protein 1 gene (IGFBP-1) was obtained from the liver of Japanese flounder (Paralichthys olivaceus) using RACE method. The complete cDNA of P. olivaceus IGFBP-1 is 1 070 bp and its ORF includes 729 bp encoding 242 amino acid residues. The IGFBP-1 was highly homologous with IGFBP-1 gene in fish through phylogency analysis. The nucleotide sequence of IGFBP-1 from P. olivaceus shared 95% homology with that of Scophthalmus maximu, and the deduced amino acid analysis showed that the sequence similarities between P. olivaceus and 5. Maximu, Seriola quinqueradiata, Perca flavescens, Salvelinus alpines, Cyprinus carpio and Danio rerio, are 89% ,89% ,84% ,79% ,67% and 67% .respectively. Semi-quantitative RT-PCR results demonstrated that IGFBP-1 mRNA was detected in unfertilized egg, fertilized egg, embryonic and larval development, and various adult tissues. Moreover, real-time quantitative RT-PCR results showed that IGFBP-1 gene had the highest expression in adult liver and was expressed in a stage-specific manner during embryonic and larval development.%利用RACE-PCR技术,从牙鲆肝脏组织总RNA中克隆得到胰岛素样生长因子结合蛋白-1(insulin-like growth factor binding protein 1,IGFBP-1)基因的全长cDNA序列,该cDNA全长为1 070 bp,开放阅读框为729 bp,编码242个氨基酸.通过系统进化树分析,牙鲆IGFBP-1与鱼类IGFBP-1基因紧密聚为一支;通过同源性比对,牙鲆IGFBP-1基因的核苷酸序列与大菱鲆同源性最高,为95%,而其推导的氨基酸序列与其它鱼类如大菱鲆、五条(蛳)、黄金鲈、红点鲑、鲤和斑马鱼的同源性分别为89%、89%、84%、79%、67%和67%.半定量RT-PCR显示,牙鲆IGFBP-1 mRNA在未受精卵、受精卵、胚胎、仔鱼发育各阶段及成体各组织均被检测.荧光定量RT-PCR结果表明,牙鲆IGFBP-1基因在成鱼肝脏中表达量最高,且在胚胎和仔鱼发育期间具有明显的时期特异性.

  7. High-throughput evaluation of aryl hydrocarbon receptor-binding sites selected via chromatin immunoprecipitation-based screening in Hepa-1c1c7 cells stimulated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    Science.gov (United States)

    Kinehara, Masaki; Fukuda, Itsuko; Yoshida, Ken-Ichi; Ashida, Hitoshi

    2008-12-01

    Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt) and binds to DNA. It has been shown that the binding of AhR to DNA depends on the dioxin response element (DRE) and controls xenobiotic-response genes. AhR-binding DNA fragments from mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD were once enriched in a chromatin immunoprecipitation (ChIP) DNA library and screened through a high-throughput southwestern chemistry-based enzyme-linked immunosorbent assay (SW-ELISA). After screening 1700 fragments, the ChIP-SW-ELISA screening strategy allowed us to isolate 77 fragments tightly interacting with AhR in the presence of TCDD. Only 39 of the 77 fragments appeared to contain a typical DRE, indicating that in some cases the DRE was dispensable for AhR-binding, while 75 fragments were located within promoter-distal regions. Genomic mapping of the 77 fragments enabled us to estimate 121 potential AhR targets including known targets such as Cyp1A1 and Cyp1B1, but only a limited number exhibited an altered expression dependent on TCDD. This study revealed the fact that TCDD-activated AhR frequently binds to promoter-distal regions even without a DRE and is not always involved in transcriptional regulation, suggesting that within the genome DNA-binding of AhR could take place often in many regions without cis-regulatory elements and might not be a key determinant to establish its regulatory function. PMID:19282623

  8. Liver Fatty Acid Binding Protein and Obesity

    OpenAIRE

    Atshaves, B.P.; Martin, G G; Hostetler, H.A.; McIntosh, A.L.; Kier, A B; Schroeder, F.

    2010-01-01

    While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes, and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity, and metabolic syndrome. Consequently, mammals evolved fatty acid binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them f...

  9. Nickel binding sites in histone proteins

    OpenAIRE

    Zoroddu, Maria Antonietta; Peana, Massimiliano Francesco; Solinas, Costantino; Medici, Serenella

    2012-01-01

    Nickel compounds are well known as human carcinogens, though the molecular events that are responsible for this are not well understood. It has been proposed that a crucial element in the mechanism of carcinogenesis is the binding of Ni(II) ions within the cell nucleus. It is known that DNA polymer binds Ni(II) only weakly, leaving the proteins of the cell nucleus as the likely Ni(II) targets. Being histone proteins the most abundant among them, they can be considered the primary sites fo...

  10. Ice-Binding Proteins and Their Function.

    Science.gov (United States)

    Bar Dolev, Maya; Braslavsky, Ido; Davies, Peter L

    2016-06-01

    Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities. PMID:27145844

  11. Antibodies against the calcium-binding protein

    International Nuclear Information System (INIS)

    Plant microsomes contain a protein clearly related to a calcium-binding protein, calsequestrin, originally found in the sarcoplasmic reticulum of muscle cells, responsible for the rapid release and uptake of Ca2+ within the cells. The location and role of calsequestrin in plant cells is unknown. To generate monoclonal antibodies specific to plant calsequestrin, mice were immunized with a microsomal fraction from cultured cells of Streptanthus tortuosus (Brassicaceae). Two clones cross-reacted with one protein band with a molecular weight equal to that of calsequestrin (57 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. This band is able to bind 45Ca2+ and can be recognized by a polyclonal antibody against the canine cardiac muscle calsequestrin. Rabbit skeletal muscle calsequestrin cross-reacted with the plant monoclonal antibodies. The plant monoclonal antibodies generated here are specific to calsequestrin protein

  12. Signal transduction by guanine nucleotide binding proteins.

    Science.gov (United States)

    Spiegel, A M

    1987-01-01

    High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, 1-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the ras gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effectors (E). A similar function has been suggested but not proven for the ras gene products. Known G-proteins include Gs and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phosphodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. 15, 237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and Go, a G-protein of unknown function that is highly abundant in brain (Sternweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 13806-13813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins. PMID:2435586

  13. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.;

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article...

  14. Odorant-binding proteins in insects.

    Science.gov (United States)

    Zhou, Jing-Jiang

    2010-01-01

    Our understanding of the molecular and biochemical mechanisms that mediate chemoreception in insects has been greatly improved after the discovery of olfactory and taste receptor proteins. However, after 50 years of the discovery of first insect sex pheromone from the silkmoth Bombyx mori, it is still unclear how hydrophobic compounds reach the dendrites of sensory neurons in vivo across aqueous space and interact with the sensory receptors. The presence of soluble polypeptides in high concentration in the lymph of chemosensilla still poses unanswered questions. More than two decades after their discovery and despite the wealth of structural and biochemical information available, the physiological function of odorant-binding proteins (OBPs) is not well understood. Here, I review the structural properties of different subclasses of insect OBPs and their binding to pheromones and other small ligands. Finally, I discuss current ideas and models on the role of such proteins in insect chemoreception. PMID:20831949

  15. Quantifying drug-protein binding in vivo

    International Nuclear Information System (INIS)

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS

  16. Maintaining cholesterol homeostasis:Sterol regulatory element-binding proteins

    Institute of Scientific and Technical Information of China (English)

    Lutz W. Weber; Meinrad Boll; Andreas Stampfl

    2004-01-01

    The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane proteins are members of the basic helix-loop-helix-leucine zipper (bHLHZip) family of transcription factors. They activate the expression of at least 30 genes involved in the synthesis of cholesterol and lipids. SREBPs are synthesized as precursor proteins in the endoplasmic reticulum (ER), where they form a complex with another protein, SREBP cleavage activating protein (SCAP).The SCAP molecule contains a sterol sensory domain. In the presence of high cellular sterol concentrations SCAP confines SREBP to the ER. With low cellular concentrations, SCAP escorts SREBP to activation in the Golgi. There, SREBP undergoes two proteolytic cleavage steps to release the mature, biologically active transcription factor, nuclear SREBP (nSREBP). nSREBP translocates to the nucleus and binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Additional transcription factors are required to activate transcription of these genes. Three different SREBPs are known, SREBPs-1a, -1c and -2. SREBP-1a and -1c are isoforms produced from a single gene by alternate splicing. SREBP-2is encoded by a different gene and does not display any isoforms. It appears that SREBPs alone, in the sequence described above, can exert complete control over cholesterol synthesis, whereas many additional factors (hormones,cytokines, etc.) are required for complete control of lipid metabolism. Medicinal manipulation of the SREBP/SCAP system is expected to prove highly beneficial in the management of cholesterol-related disease.

  17. Brain hyaluronan binding protein inhibits tumor growth

    Institute of Scientific and Technical Information of China (English)

    高锋; 曹曼林; 王蕾

    2004-01-01

    Background Great efforts have been made to search for the angiogenic inhibitors in avascular tissues. Several proteins isolated from cartilage have been proved to have anti-angiogenic or anti-tumour effects. Because cartilage contains a great amount of hyaluronic acid (HA) oligosaccharides and abundant HA binding proteins (HABP), therefore, we speculated that HABP might be one of the factors regulating vascularization in cartilage or anti-angiogenesis in tumours. The purpose of this research was to evaluale the effects of hyaluronan binding protein on inhibiting tumour growth both in vivo and vitro. Methods A unique protein termed human brain hyaluronan (HA) binding protein (b-HABP) was cloned from human brain cDNA library. MDA-435 human breast cancer cell line was chosen as a transfectant. The in vitro underlying mechanisms were investigated by determining the possibilities of MDA-435/b-HABP colony formation on soft agar, the effects of the transfectant on the proliferation of endothelial cells and the expression levels of caspase 3 and FasL from MDA-435/b-HABP. The in vivo study included tumour growth on the chorioallantoic membrane (CAM) of chicken embryos and nude mice. Results Colony formation assay revealed that the colonies formed by MDA-435/b-HABP were greatly reduced compared to mock transfectants. The conditioned media from MDA-435/b-HABP inhibited the growth of endothelial cells in culture. Caspase 3 and FasL expressions were induced by MDA-435/b-HABP. The size of tumours of MDA-435/b-HABP in both CAM and nude mice was much smaller than that of MDA-435 alone. Conclusions Human brain hyaluronan binding protein (b-HABP) may represent a new kind of naturally existing anti-tumour substance. This brain-derived glycoprotein may block tumour growth by inducing apoptosis of cancer cells or by decreasing angiogenesis in tumour tissue via inhibiting proliferation of endothelial cells.

  18. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert; Rydström, Jan

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  19. Sulforaphane induces CYP1A1 mRNA, protein, and catalytic activity levels via an AhR-dependent pathway in murine hepatoma Hepa 1c1c7 and human HepG2 cells.

    Science.gov (United States)

    Anwar-Mohamed, Anwar; El-Kadi, Ayman O S

    2009-03-01

    Recent reports have proposed that some naturally occurring phytochemicals can function as anticancer agents mainly through inducing phase II drug detoxification enzymes. Of these phytochemicals, isothiocyanates sulforaphane (SUL), present in broccoli, is by far the most extensively studied. In spite of its positive effect on phase II drug metabolizing enzymes, its effect on the phase I bioactivating enzyme cytochrome P450 1a1 (Cyp1a1) is still a matter of debate. As a first step to investigate this effect, Hepa 1c1c7 and HepG2 cells were treated with various concentration of SUL. Our results showed that SUL-induced CYP1A1 mRNA in a dose- and time-dependent manner. Furthermore, this induction was further reflected on the protein and catalytic activity levels. Investigating the effect of SUL at the transcriptional level revealed that SUL increases the Cyp1a1 mRNA as early as 1h. The RNA polymerase inhibitor actinomycin D (Act-D) completely abolished the SUL-induced Cyp1a1 mRNA. Furthermore, SUL successfully activated AhR transformation and its subsequent binding to the XRE. At the post-transcriptional level, SUL did not affect the levels of existing Cyp1a1 mRNA transcripts. This is the first demonstration that the broccoli-derived SUL can directly induce Cyp1a1 gene expression in an AhR-dependent manner and represents a novel mechanism by which SUL induces this enzyme. PMID:19013013

  20. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    OpenAIRE

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesi...

  1. Where metal ions bind in proteins.

    OpenAIRE

    Yamashita, M M; Wesson, L.; Eisenman, G.; Eisenberg, D.

    1990-01-01

    The environments of metal ions (Li+, Na+, K+, Ag+, Cs+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+) in proteins and other metal-host molecules have been examined. Regardless of the metal and its precise pattern of ligation to the protein, there is a common qualitative feature to the binding site: the metal is ligated by a shell of hydrophilic atomic groups (containing oxygen, nitrogen, or sulfur atoms) and this hydrophilic shell is embedded within a larger shell of hydrophobic atomic groups (containing car...

  2. Triple NF-kB binding sites and LTR sequence similarities in HIV-1C isolates irrespective of helminth co-infection

    Science.gov (United States)

    2014-01-01

    Background Helminth infections as well as structural alternations in the long-terminal repeat (LTR) regions of HIV-1 are known to contribute to elevated HIV RNA level and enhance HIV-1 diseases progression. However, the impact of helminths infections on the occurrences of triple NF-κB and genetic variability in LTR region of HIV-1C isolates is not known. We aimed to examine the presence of genetic variability in the LTR region of HIV-1C isolates during chronic HIV-helminth co-infection. Methods HIV-1C infected Ethiopians with (n = 22) and without (n = 20) helminth infection were included. The LTR region of HIV was amplified and sequenced. Sequences were aligned with reference set from the Los Alamos HIV database. Phylogenetic analysis and frequency of polymorphic changes was performed by the neighbour-joining method using Geneious Basic software. Results All LTR sequences from patients with or without of helminth co-infection clustered with HIV-1 subtype C with two distinct subclusters (C and C’). The enhancer element was found to have three copies of 10-base pair binding sites for NF-κBs which is an evidence for predominance of triple NF-κB sites (94%) in HIV-1C isolates irrespective of helminths co-infection and subclusters. Moreover, irrespective of helminth co-infection and C/C’ subclusters high sequences similarity in LTR was observed. There was no significant difference in plasma HIV RNA level between HIV-1 C and C’ subclusters. Conclusions Despite the small sample size, the predominance of triple NF-κB binding sites and high sequence similarities in LTR region irrespective of helminths infection suggest the natural occurrence of the three NF-κB binding sites in HIV-1C isolates without the influence of secondary infection. Thus, the higher HIV-1C viraemia in helminth co-infected individuals is more likely a result of immune activation rather than LTR sequence variation. Moreover, the lack of significant difference in plasma HIV RNA level

  3. Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

    International Nuclear Information System (INIS)

    Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells

  4. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  5. Protein and ligand adaptation in a retinoic acid binding protein.

    OpenAIRE

    Pattanayek, R.; Newcomer, M E

    1999-01-01

    A retinoic acid binding protein isolated from the lumen of the rat epididymis (ERABP) is a member of the lipocalin superfamily. ERABP binds both the all-trans and 9-cis isomers of retinoic acid, as well as the synthetic retinoid (E)-4-[2-(5,6,7,8)-tetrahydro-5,5,8,8-tetramethyl-2 napthalenyl-1 propenyl]-benzoic acid (TTNPB), a structural analog of all-trans retinoic acid. The structure of ERABP with a mixture of all-trans and 9-cis retinoic acid has previously been reported. To elucidate any ...

  6. Dissection of the Critical Binding Determinants of Cellular Retinoic Acid Binding Protein II by Mutagenesis and Fluorescence Binding Assay

    OpenAIRE

    Vasileiou, Chrysoula; Lee, Kin Sing Stephen; Crist, Rachael M.; Vaezeslami, Soheila; Goins, Sarah M.; Geiger, James H.; Borhan, Babak

    2009-01-01

    The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by ...

  7. Landscape of protein-small ligand binding modes.

    Science.gov (United States)

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them. PMID:27327045

  8. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  9. Alternative polyadenylation and RNA-binding proteins.

    Science.gov (United States)

    Erson-Bensan, Ayse Elif

    2016-08-01

    Our understanding of the extent of microRNA-based gene regulation has expanded in an impressive pace over the past decade. Now, we are beginning to better appreciate the role of 3'-UTR (untranslated region) cis-elements which harbor not only microRNA but also RNA-binding protein (RBP) binding sites that have significant effect on the stability and translational rate of mRNAs. To add further complexity, alternative polyadenylation (APA) emerges as a widespread mechanism to regulate gene expression by producing shorter or longer mRNA isoforms that differ in the length of their 3'-UTRs or even coding sequences. Resulting shorter mRNA isoforms generally lack cis-elements where trans-acting factors bind, and hence are differentially regulated compared with the longer isoforms. This review focuses on the RBPs involved in APA regulation and their action mechanisms on APA-generated isoforms. A better understanding of the complex interactions between APA and RBPs is promising for mechanistic and clinical implications including biomarker discovery and new therapeutic approaches. PMID:27208003

  10. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    Science.gov (United States)

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  11. HPF1/C4orf27 Is a PARP-1-Interacting Protein that Regulates PARP-1 ADP-Ribosylation Activity

    OpenAIRE

    Gibbs-Seymour, Ian; Fontana, Pietro; Rack, Johannes Gregor Matthias; Ahel, Ivan

    2016-01-01

    Summary We report the identification of histone PARylation factor 1 (HPF1; also known as C4orf27) as a regulator of ADP-ribosylation signaling in the DNA damage response. HPF1/C4orf27 forms a robust protein complex with PARP-1 in cells and is recruited to DNA lesions in a PARP-1-dependent manner, but independently of PARP-1 catalytic ADP-ribosylation activity. Functionally, HPF1 promotes PARP-1-dependent in trans ADP-ribosylation of histones and limits DNA damage-induced hyper-automodificatio...

  12. Isolation of a Thiamine-binding Protein from Rice Germ and Distribution of Similar Proteins.

    Science.gov (United States)

    Shimizu, M; Yoshida, T; Toda, T; Iwashima, A; Mitsunaga, T

    1996-01-01

    A thiamine-binding protein was purified from rice germ (Oryza sativa L.) by extraction, salting-out with ammonium sulfate, and column chromatography. From the results of molecular mass, Kd and Bmax values for thiamine-binding, binding specificity for thiamine phosphates and analog, the protein was suggested to be identical to the thiamine-binding protein in rice bran. The thiamine-binding protein w as more efficiently purified from rice germ than from rice bran. The protein was rich in glutamic acid (and/or glutamine) and glycine. The protein did not show immunological similarity to thiamine-binding proteins in buckwheat and sesame seeds. However proteins similar to the thiamine-binding protein from rice germ existed in gramineous seeds. They were suggested to have thiamine-binding activity and to be of the same molecular mass as the thiamine-binding protein. PMID:27299548

  13. Identification of Treponema pallidum penicillin-binding proteins.

    OpenAIRE

    Cunningham, T M; Miller, J N; Lovett, M A

    1987-01-01

    Penicillin-binding proteins of 180, 89, 80, 68, 61, 41, and 38 kilodaltons were identified in Treponema pallidum (Nichols) by their covalent binding of [35S]benzylpenicillin. Penicillin-binding proteins are localized in the plasma membranes of many bacterial species and may serve as useful markers for determining plasma membrane intactness in T. pallidum fractionation studies.

  14. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    OpenAIRE

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associa...

  15. Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

    Directory of Open Access Journals (Sweden)

    Sowmya Sampath

    Full Text Available Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP and the Duffy Antigen Receptor for Chemokines (DARC and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

  16. STRUCTURAL FEATURES OF PLANT CHITINASES AND CHITIN-BINDING PROTEINS

    NARCIS (Netherlands)

    BEINTEMA, JJ

    1994-01-01

    Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains,of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now,

  17. Cobalamin and folate binding proteins in human tumour tissue.

    OpenAIRE

    Sheppard, K; Bradbury, D A; Davies, J. M.; Ryrie, D. R.

    1984-01-01

    The serum of an 84 year old man with disseminated carcinoma was found to contain extremely high concentrations of cobalamin and of a cobalamin binding protein with trans-cobalamin I characteristics. Tumour tissue samples obtained at necropsy contained considerably higher concentrations of cobalamin binding protein (R-binder) than normal tissues. Tumour tissues also contained increased concentrations of specific folate binding protein. In all tissues studied a close correlation existed between...

  18. Minimalistic predictor of protein binding energy: contribution of solvation factor to protein binding.

    Science.gov (United States)

    Choi, Jeong-Mo; Serohijos, Adrian W R; Murphy, Sean; Lucarelli, Dennis; Lofranco, Leo L; Feldman, Andrew; Shakhnovich, Eugene I

    2015-02-17

    It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software. PMID:25692584

  19. Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

    OpenAIRE

    Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.

    2012-01-01

    Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the p...

  20. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins

    Directory of Open Access Journals (Sweden)

    Elisa E. Figueroa-Angulo

    2015-11-01

    Full Text Available Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs that interact with an iron responsive element (IRE located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

  1. Inhibition of tristetraprolin deadenylation by poly(A) binding protein

    OpenAIRE

    Rowlett, Robert M.; Chrestensen, Carol A.; Schroeder, Melanie J.; Harp, Mary G.; Pelo, Jared W.; Shabanowitz, Jeffery; DeRose, Robert; Hunt, Donald F.; Sturgill, Thomas W.; Worthington, Mark T.

    2008-01-01

    Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevente...

  2. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  3. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  4. Rapid determination of thyroxine binding proteins of human serum

    Directory of Open Access Journals (Sweden)

    Arima,Terukatsu

    1976-02-01

    Full Text Available A simple method is described for determing thyroxine binding proteins in human serum by electrophoresis at pH 8.6, using cellulose acetate membrane as the supporting medium. The procedure had high reliability in sera of normal subjects, pregnant women and patients with decreased thyroxine binding capacity of thyroxine binding globulin.

  5. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B;

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  6. Calmodulin Binding Proteins and Alzheimer’s Disease

    Science.gov (United States)

    O’Day, Danton H.; Eshak, Kristeen; Myre, Michael A.

    2015-01-01

    Abstract The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer’s disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer’s disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer’s disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  7. SCOWLP classification: Structural comparison and analysis of protein binding regions

    Directory of Open Access Journals (Sweden)

    Anders Gerd

    2008-01-01

    Full Text Available Abstract Background Detailed information about protein interactions is critical for our understanding of the principles governing protein recognition mechanisms. The structures of many proteins have been experimentally determined in complex with different ligands bound either in the same or different binding regions. Thus, the structural interactome requires the development of tools to classify protein binding regions. A proper classification may provide a general view of the regions that a protein uses to bind others and also facilitate a detailed comparative analysis of the interacting information for specific protein binding regions at atomic level. Such classification might be of potential use for deciphering protein interaction networks, understanding protein function, rational engineering and design. Description Protein binding regions (PBRs might be ideally described as well-defined separated regions that share no interacting residues one another. However, PBRs are often irregular, discontinuous and can share a wide range of interacting residues among them. The criteria to define an individual binding region can be often arbitrary and may differ from other binding regions within a protein family. Therefore, the rational behind protein interface classification should aim to fulfil the requirements of the analysis to be performed. We extract detailed interaction information of protein domains, peptides and interfacial solvent from the SCOWLP database and we classify the PBRs of each domain family. For this purpose, we define a similarity index based on the overlapping of interacting residues mapped in pair-wise structural alignments. We perform our classification with agglomerative hierarchical clustering using the complete-linkage method. Our classification is calculated at different similarity cut-offs to allow flexibility in the analysis of PBRs, feature especially interesting for those protein families with conflictive binding regions

  8. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    OpenAIRE

    Selvaraj S; Jayaram B; Saranya N; Gromiha M; Fukui Kazuhiko

    2011-01-01

    Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such...

  9. Clinical relevance of drug binding to plasma proteins

    Science.gov (United States)

    Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

    2014-12-01

    Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

  10. The actin binding protein adseverin regulates osteoclastogenesis.

    Science.gov (United States)

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  11. The actin binding protein adseverin regulates osteoclastogenesis.

    Directory of Open Access Journals (Sweden)

    Siavash Hassanpour

    Full Text Available Adseverin (Ads, a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG. Ads is induced during OCG downstream of RANK-ligand (RANKL stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion.

  12. Concentration-dependent Cu(II) binding to prion protein

    Science.gov (United States)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2008-03-01

    The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

  13. Pumilio Puf domain RNA-binding proteins in Arabidopsis

    OpenAIRE

    Abbasi, Nazia; Park, Youn-Il; Choi, Sang-Bong

    2011-01-01

    Pumilio proteins are a class of RNA-binding proteins harboring Puf domains (or PUM-HD; Pumilio-Homology Domain), named after the founding members, Pumilio (from Drosophila melanogaster) and FBF (Fem-3 mRNA-Binding Factor from Caenorhabditis elegans). The domains contain multiple tandem repeats each of which recognizes one RNA base and is comprised of 35–39 amino acids. Puf domain proteins have been reported in organisms ranging from single-celled yeast to higher multicellular eukaryotes, such...

  14. Grafting odorant binding proteins on diamond bio-MEMS

    OpenAIRE

    Manai, Raafa; Scorsone, E.; Rousseau, L.; Ghassemi, F.; Possas Abreu, M.; Lissorgues, G.; Tremillon, N.; Ginisty, H; Arnault, J.-C.; Tuccori, E.; Bernabei, M.; Cali, K.; Persaud, K C; Bergonzo, P.

    2014-01-01

    Odorant binding proteins (OBPs) are small soluble proteins found in olfactory systems that are capable of binding several types of odorant molecules. Cantilevers based on polycrystalline diamond surfaces are very promising as chemical transducers. Here two methods were investigated for chemically grafting porcine OBPs on polycrystalline diamond surfaces for biosensor development. The first approach resulted in random orientation of the immobilized proteins over the surface. The second approac...

  15. The clinical significance of fatty acid binding proteins

    OpenAIRE

    Barbara Choromańska; Piotr Myśliwiec; Jacek Dadan; Hady Razak Hady; Adrian Chabowski

    2011-01-01

    Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs) that bind long-chain fatty acids (LCFA), and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum). So far, nine types of these proteins have been described, and their name refers to the place in which they were first ...

  16. Cooperative binding modes of Cu(II) in prion protein

    Science.gov (United States)

    Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

    2007-03-01

    The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

  17. Thermodynamic parameters of the binding of retinol to binding proteins and to membranes

    International Nuclear Information System (INIS)

    Retinol (vitamin A alcohol) is a hydrophobic compound and distributes in vivo mainly between binding proteins and cellular membranes. To better clarify the nature of the interactions of retinol with these phases which have a high affinity for it, the thermodynamic parameters of these interactions were studied. The temperature-dependence profiles of the binding of retinol to bovine retinol binding protein, bovine serum albumin, unilamellar vesicles of dioleoylphosphatidylcholine, and plasma membranes from rat liver were determined. It was found that binding of retinol to retinol binding protein is characterized by a large increase in entropy and no change in enthalpy. Binding to albumin is driven by enthalpy and is accompanied by a decrease in entropy. Partitioning of retinal into unilamellar vesicles and into plasma membranes is stabilized both by enthalpic and by entropic components. The implications of these finding are discussed

  18. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Directory of Open Access Journals (Sweden)

    Selvaraj S

    2011-10-01

    Full Text Available Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. Results We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. Conclusions The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

  19. Stereoselective binding of chiral drugs to plasma proteins

    Institute of Scientific and Technical Information of China (English)

    Qi SHEN; Lu WANG; Hui ZHOU; Hui-di JIANG; Lu-shan YU; Su ZENG

    2013-01-01

    Chiral drugs show distinct biochemical and pharmacological behaviors in the human body.The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity,which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles.In this review,the stereoselective binding of chiral drugs to human serum albumin (HSA),α1-acid glycoprotein (AGP)and lipoprotein,three most important proteins in human plasma,are detailed.Furthermore,the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed.Apart from the stereoselectivity of enantiomer-protein binding,enantiomer-enantiomer interactions that may induce allosteric effects are also described.Additionally,the techniques and methods used to determine drug-protein binding parameters are briefly reviewed.

  20. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins.

    Science.gov (United States)

    Zamore, P D; Williamson, J R; Lehmann, R

    1997-01-01

    Translation of hunchback(mat) (hb[mat]) mRNA must be repressed in the posterior of the pre-blastoderm Drosophila embryo to permit formation of abdominal segments. This translational repression requires two copies of the Nanos Response Element (NRE), a 16-nt sequence in the hb[mat] 3' untranslated region. Translational repression also requires the action of two proteins: Pumilio (PUM), a sequence-specific RNA-binding protein; and Nanos, a protein that determines the location of repression. Binding of PUM to the NRE is thought to target hb(mat) mRNA for repression. Here, we show the RNA-binding domain of PUM to be an evolutionarily conserved, 334-amino acid region at the carboxy-terminus of the approximately 158-kDa PUM protein. This contiguous region of PUM retains the RNA-binding specificity of full-length PUM protein. Proteins with sequences homologous to the PUM RNA-binding domain are found in animals, plants, and fungi. The high degree of sequence conservation of the PUM RNA-binding domain in other far-flung species suggests that the domain is an ancient protein motif, and we show that conservation of sequence reflects conservation of function: that is, the homologous region from a human protein binds RNA with sequence specificity related to but distinct from Drosophila PUM. PMID:9404893

  1. Guardian of Genetic Messenger-RNA-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Antje Anji

    2016-01-01

    Full Text Available RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

  2. Convergent evolution among immunoglobulin G-binding bacterial proteins.

    OpenAIRE

    Frick, I M; Wikström, M.; Forsén, S.; Drakenberg, T; Gomi, H.; Sjöbring, U; Björck, L

    1992-01-01

    Protein G, a bacterial cell-wall protein with high affinity for the constant region of IgG (IgGFc) antibodies, contains homologous repeats responsible for the interaction with IgGFc. A synthetic peptide corresponding to an 11-amino acid-long sequence in the COOH-terminal region of the repeats was found to bind to IgGFc and block the interaction with protein G. Moreover, two other IgGFc-binding bacterial proteins (proteins A and H), which do not contain any sequences homologous to the peptide,...

  3. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

    [3H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [3H]-cocaine with a high affinity site of 170 fmole/mg protein. The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [3H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear

  4. Electrochemistry of heparin binding to tau protein on Au surfaces

    International Nuclear Information System (INIS)

    Highlights: • Anionic heparin binds tau protein film on Au • N-terminal of tau protein is critical for heparin binding • Negatively charged heparin binds positively charged tau domains • Heparin binding to tau increases charge transfer resistance - ABSTRACT: The tau protein is a neurodegenerative disease biomarker. The in vitro aggregation of tau is triggered by electrostatic charge imbalance induced by an anionic inducing agent, such as heparin. The binding of the tau-heparin complex is based on electrostatic interactions, but the exact binding mode of heparin to the tau protein has not been fully identified. In this work, the effects of the tau protein orientation on gold (Au) electrode to heparin were explored by the cyclic voltammetry and electrochemical impedance spectroscopy. To modulate the accessibility of N-terminal of the tau to heparin, the tau films on Au surfaces were fabricated in two ways: immobilization of tau via the N-terminal of tau protein (N-tau-Au) or by the Cys291/Cys322 residues, located in the R-repeat domains of the tau protein (Cys-tau-Au). The sulfur-Au bonding was characterized by X-ray photoelectron spectroscopy. The charge transfer resistance was measured for N-tau-Au and Cys-tau-Au as a function of heparin concentration. The heparin concentration range was varied from 0.2 pM to 216 μM with the optimal binding concentration at 21 nM (the highest charge transfer resistance value). The heparin binding to tau films was investigated in the presence of [Fe(CN)6]3−/4− or benzoquinone redox probes. The tau-heparin binding was greater for the Cys-tau-Au surface over N-tau-Au, indicating specific tau domains may be required for optimal heparin binding

  5. Hepatitis B Virus X Protein Up-Regulates AKR1C1 Expression Through Nuclear Factor-Y in Human Hepatocarcinoma Cells

    OpenAIRE

    Kai LI; Ding, Shijia; Chen, Ke; Qin, Dongdong; Qu, Jialin; Wang, Sen; Sheng, Yanrui; Zou, Chengcheng; Chen, Limin; Tang, Hua

    2013-01-01

    Background The hepatitis B virus X (HBx) protein has long been recognized as an important transcriptional transactivator of several genes. Human aldo-keto reductase family 1, member C1 (AKR1C1), a member of the family of AKR1CS, is significantly increased in HBx-expressed cells. Objectives This study aimed to investigate the possible mechanism of HBx in regulating AKR1C1 expression in HepG2.2.15 cells and the role of AKR1C1 for HBV-induced HCC. Materials and Methods RT-PCR was performed to de...

  6. Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S;

    1992-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous...... remarkable correspondence between the structural modules of ACBP/DBI as determined by 1H nuclear magnetic resonance spectroscopy and the exon-intron architecture of the ACBP/DBI gene. Detailed analyses of transcription of the ACBP/DBI gene in brain and liver were performed to map transcription initiation...

  7. Drosophila Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity

    Directory of Open Access Journals (Sweden)

    Kirchner Jasmin

    2008-04-01

    Full Text Available Abstract Background Protein phosphatase 1 (PP1 is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c fall into PP1α and PP1β subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1α and PP1β. Results URI (unconventional prefoldin RPB5 interactor is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that Drosophila Uri binds PP1α with much higher affinity than PP1β, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a uri loss of function allele, and show that uri is essential for viability in Drosophila. uri mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei. Conclusion Uri is the first PP1α specific binding protein to be described in Drosophila. Uri protein plays a role in transcriptional regulation. Activity of uri is required to maintain DNA integrity and cell survival in normal development.

  8. Niobium Uptake and Release by Bacterial Ferric Ion Binding Protein

    Directory of Open Access Journals (Sweden)

    Yanbo Shi

    2010-01-01

    Full Text Available Ferric ion binding proteins (Fbps transport FeIII across the periplasm and are vital for the virulence of many Gram negative bacteria. Iron(III is tightly bound in a hinged binding cleft with octahedral coordination geometry involving binding to protein side chains (including tyrosinate residues together with a synergistic anion such as phosphate. Niobium compounds are of interest for their potential biological activity, which has been little explored. We have studied the binding of cyclopentadienyl and nitrilotriacetato NbV complexes to the Fbp from Neisseria gonorrhoeae by UV-vis spectroscopy, chromatography, ICP-OES, mass spectrometry, and Nb K-edge X-ray absorption spectroscopy. These data suggest that NbV binds strongly to Fbp and that a dinuclear NbV centre can be readily accommodated in the interdomain binding cleft. The possibility of designing niobium-based antibiotics which block iron uptake by pathogenic bacteria is discussed.

  9. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovir

  10. Expected and unexpected features of protein-binding RNA aptamers

    DEFF Research Database (Denmark)

    Bjerregaard, Nils; Andreasen, Peter A; Dupont, Daniel M

    2016-01-01

    RNA molecules with high affinity to specific proteins can be isolated from libraries of up to 10(16) different RNA sequences by systematic evolution of ligands by exponential enrichment (SELEX). These so-called protein-binding RNA aptamers are often interesting, e.g., as modulators of protein...... function for therapeutic use, for probing the conformations of proteins, for studies of basic aspects of nucleic acid-protein interactions, etc. Studies on the interactions between RNA aptamers and proteins display a number of expected and unexpected features, including the chemical nature of the...... interacting RNA-protein surfaces, the conformation of protein-bound aptamer versus free aptamer, the conformation of aptamer-bound protein versus free protein, and the effects of aptamers on protein function. Here, we review current insights into the details of RNA aptamer-protein interactions. For further...

  11. The interrelationship between ligand binding and self-association of the folate binding protein

    DEFF Research Database (Denmark)

    Holm, Jan; Schou, Christian; Babol, Linnea N.;

    2011-01-01

    The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of...

  12. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    Science.gov (United States)

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  13. Autoinhibition of Mint1 adaptor protein regulates amyloid precursor protein binding and processing

    OpenAIRE

    Matos, Maria F.; Xu, Yibin; Dulubova, Irina; Otwinowski, Zbyszek; Richardson, John M.; Tomchick, Diana R.; Rizo, Josep; Ho, Angela

    2012-01-01

    Mint adaptor proteins bind to the amyloid precursor protein (APP) and regulate APP processing associated with Alzheimer’s disease; however, the molecular mechanisms underlying Mint regulation in APP binding and processing remain unclear. Biochemical, biophysical, and cellular experiments now show that the Mint1 phosphotyrosine binding (PTB) domain that binds to APP is intramolecularly inhibited by the adjacent C-terminal linker region. The crystal structure of a C-terminally extended Mint1 PT...

  14. Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

    Science.gov (United States)

    Mikkelsen, R B; Wallach, D F

    1976-05-21

    (1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone. PMID:6061

  15. Identification of lectin-binding proteins in Chlamydia species.

    OpenAIRE

    Swanson, A F; Kuo, C. C.

    1990-01-01

    Lectin-binding proteins of chlamydiae were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. All three Chlamydia species tested expressed two proteins when whole-elementary-body lysates were reacted with the biotinylated lectin Dolichos biflorus agglutinin. The protein with a molecular mass of 18 kilodaltons (kDa) responded strongly compared with a higher-molecular-mass protein that varied from 27 to 32 kDa with each chlamydia strain tested. Among six l...

  16. Optimizing a coarse-grained model for the recognition of protein-protein binding

    OpenAIRE

    Emperador, Agustí; Orozco, Modesto

    2015-01-01

    We are optimizing a force-field to be used with our coarsegrained protein model for the recognition of protein -protein binding. We have found that, apart from ranking correctly the ligand-receptor conformations generated in a protein-protein docking algorithm, our model is able to distinguish binding (experimental structure) from nonbinding (false positive) conformations for many complexes. This suggests us that the model could have a good performance in complete cross-d...

  17. PRELIMINARY STUDY OF EXTRACTABLE PROTEIN BINDING USING MALEIC ANHYDRIDE COPOLYMER

    Institute of Scientific and Technical Information of China (English)

    Thirawan Nipithakul; Ladawan Watthanachote; Nanticha Kalapat

    2012-01-01

    A preliminary study of using maleic anhydride copolymer for protein binding has been carried out.The polymeric films were prepared by compression of the purified resin and annealing the film to induce efficient back formation of the anhydride groups.The properties of the film surface were analyzed by attenuated total reflection Fourier transforms infrared spectroscopy and water contact angle measurements.The protein content was determined by Bradford assay.To obtain optimum conditions,immersion time for protein binding was examined.Results revealed that proteins can be successfully immobilized onto the film surface via covalent linkage.The efficiency of the covalent binding of the extractable protein to maleic anhydride-polyethylene film was estimated at 69.87 μtg/cm2,although the film had low anhydride content (3%) on the surface.

  18. Binding of CCAAT displacement protein CDP to adenovirus packaging sequences.

    Science.gov (United States)

    Erturk, Ece; Ostapchuk, Philomena; Wells, Susanne I; Yang, Jihong; Gregg, Keqin; Nepveu, Alain; Dudley, Jaquelin P; Hearing, Patrick

    2003-06-01

    Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis-acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding activities to Ad DNA packaging. The results of these experiments reveal that COUP-TF and Oct-1 binding does not play a functional role in Ad packaging, whereas P-complex binding directly correlates with packaging function. We demonstrate that P complex contains the cellular protein CCAAT displacement protein (CDP) and that full-length CDP is found in purified virus particles. In addition to cellular factors, previous evidence indicates that viral factors play a role in the initiation of viral DNA packaging. We propose that CDP, in conjunction with one or more viral proteins, binds to the packaging sequences of Ad to initiate the encapsidation process. PMID:12743282

  19. Characterization of cap binding proteins associated with the nucleus

    International Nuclear Information System (INIS)

    Eucaryotic mRNAs a carry 7-methylguanosine triphosphate residue (called cap structure) at their 5' terminus. The cap plays an important role in RNA recognition. Cap binding proteins (CBP) of HeLa cells were identified by photoaffinity labelling using the cap analogue γ-(32P)-(4-(benzoyl-phenyl)methylamido)-7-methylguanosine-5'-triphosphate (BP-m7GTP). Photoreaction of this cap analogue with HeLa cell initiation factors resulted in specific labelling of two polypeptides of Msub(r) 37000 and 26000. The latter was also labelled in crude initiation factors prepared from reticulocytes and is identical to the cap binding protein CBP I previously identified. These cap binding proteins were also affinity labelled in poliovirus infected cell extracts. Photoaffinity reaction with BP-m7GTP of whole HeLa cell homogenate showed three additional polypeptides with Msub(r) 120000, 89000 and 80000. These cap binding proteins were found to be associated with the nucleus and are therefore referred to as nuclear cap binding proteins, i.e. NCBP 1, NCBP 2 and NCBP 3. They were also present in splicing extracts. Photoaffinity labelling in these nuclear extracts was differentially inhibited by various cap analogues and capped mRNAs. Affinity chromatography on immobilized globin mRNA led to a partial separation of the three nuclear cap binding proteins. Chromatography on m7GTP-Sepharose resulted in a specific binding of NCBP 3. The different behaviour of the cap binding proteins suggests that they are functionally distinct and that they might be involved in different processes requiring cap recognition. (Author)

  20. High-throughput analysis of protein-DNA binding affinity.

    Science.gov (United States)

    Franco-Zorrilla, José M; Solano, Roberto

    2014-01-01

    Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro. PMID:24057393

  1. High-Fidelity DNA Sensing by Protein Binding Fluctuations

    CERN Document Server

    Tlusty, Tsvi; Libchaber, Albert; 10.1103/PhysRevLett.93.258103

    2010-01-01

    One of the major functions of RecA protein in the cell is to bind single-stranded DNA exposed upon damage, thereby triggering the SOS repair response.We present fluorescence anisotropy measurements at the binding onset, showing enhanced DNA length discrimination induced by adenosine triphosphate consumption. Our model explains the observed DNA length sensing as an outcome of out-of equilibrium binding fluctuations, reminiscent of microtubule dynamic instability. The cascade architecture of the binding fluctuations is a generalization of the kinetic proofreading mechanism. Enhancement of precision by an irreversible multistage pathway is a possible design principle in the noisy biological environment.

  2. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus

    2005-01-01

    ubiquitin conjugation to endoplasmic reticulum degradation), UEV [ubiquitin E2 (ubiquitin-conjugating enzyme) variant] and NZF (nuclear protein localization gene 4 zinc finger) domain-containing proteins appear to have more specialized functions. Here we discuss functional and structural properties of...

  3. Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine

    International Nuclear Information System (INIS)

    Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 125I-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although 125I-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase A2 treatment of dipalmitoylphosphatidylcholine. 125I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 mM EGTA into the binding buffer reduced much of the 125I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed

  4. Lipid A binding proteins in macrophages detected by ligand blotting

    International Nuclear Information System (INIS)

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with 32P/sub i/ (109 dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with [32P]-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS

  5. Mining the characteristic interaction patterns on protein-protein binding interfaces.

    Science.gov (United States)

    Li, Yan; Liu, Zhihai; Han, Li; Li, Chengke; Wang, Renxiao

    2013-09-23

    Protein-protein interactions are observed in various biological processes. They are important for understanding the underlying molecular mechanisms and can be potential targets for developing small-molecule regulators of such processes. Previous studies suggest that certain residues on protein-protein binding interfaces are "hot spots". As an extension to this concept, we have developed a residue-based method to identify the characteristic interaction patterns (CIPs) on protein-protein binding interfaces, in which each pattern is a cluster of four contacting residues. Systematic analysis was conducted on a nonredundant set of 1,222 protein-protein binding interfaces selected out of the entire Protein Data Bank. Favored interaction patterns across different protein-protein binding interfaces were retrieved by considering both geometrical and chemical conservations. As demonstrated on two test tests, our method was able to predict hot spot residues on protein-protein binding interfaces with good recall scores and acceptable precision scores. By analyzing the function annotations and the evolutionary tree of the protein-protein complexes in our data set, we also observed that protein-protein interfaces sharing common characteristic interaction patterns are normally associated with identical or similar biological functions. PMID:23930922

  6. The cobalamin-binding protein in zebrafish is an intermediate between the three cobalamin-binding proteins in human.

    Directory of Open Access Journals (Sweden)

    Eva Greibe

    Full Text Available In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC, intrinsic factor (IF, and haptocorrin (HC, are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish and ambient water (13.5 pmol/fish associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin (like human IF, but unlike human HC and TC. The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared with human IF. The absorbance spectrum of the purified protein in complex with hydroxo-cobalamin resembled those of human HC and IF, but not TC. We searched available databases to further explore the phylogenies of the three cobalamin-binding proteins in higher vertebrates. Apparently, TC-like proteins are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals. Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis about a common ancestral gene for all cobalamin-binding proteins in higher vertebrates.

  7. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T;

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...... by a simple two step protocol combining ion exchange chromatography and gel filtration. Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid and the peroxisomal proliferator bezafibrate to L-FABP have been determined by titration calorimetry. All ligands were...... bound in a 2:1 stoichiometry, the dissociation constants for the first ligand bound were all in the micro molar range. Oleic acid was bound with the highest affinity and a Kd of 0.26 microM. Furthermore, binding of cholesterol to L-FABP was investigated with the Lipidex assay, a liposome binding assay...

  8. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E; Krogsdam, A M; Jorgensen, H F;

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...

  9. Solid-binding Proteins for Modification of Inorganic Substrates

    Science.gov (United States)

    Coyle, Brandon Laurence

    Robust and simple strategies to directly functionalize graphene- and diamond-based nanostructures with proteins are of considerable interest for biologically driven manufacturing, biosensing and bioimaging. In this work, we identify a new set of carbon binding peptides that vary in overall hydrophobicity and charge, and engineer two of these sequences (Car9 and Car15) within the framework of various proteins to exploit their binding ability. In addition, we conducted a detailed analysis of the mechanisms that underpin the interaction of the fusion proteins with carbon and silicon surfaces. Through these insights, we were able to develop proteins suitable for dispersing graphene flakes and carbon nanotubes in aqueous solutions, while retaining protein activity. Additionally, our investigation into the mechanisms of adhesion for our carbon binding peptides inspired a cheap, disposable protein purification system that is more than 10x cheaper than commonly used His-tag protein purification. Our results emphasize the importance of understanding both bulk and molecular recognition events when exploiting the adhesive properties of solid-binding peptides and proteins in technological applications.

  10. Zeatin-binding proteins in barley leaves

    International Nuclear Information System (INIS)

    Highly labelled tritium-zeatin was used in the work to clarify for the first time a protein factor that is present in cytokinin-sensitive vegetative organs of plants (barley leaves) and which possesses the properties of a cytokinin receptor. Aliquots of tritium-zeatin were mixed with a solution of protein and incubated for several hours in buffer. Following incubation, protein was precipitated by ammonium sulfate at 90% of saturation, and radioactivity of the precipitate was checked in a dioxane scintillator with an efficiency of about 35%. It is shown that the characteristics of interaction of the clarified specific protein sites with cytokinins in regard to a number of criteria correspond to the characteristics expected of receptors of these phytohormones

  11. Zeatin-binding proteins in barley leaves

    Energy Technology Data Exchange (ETDEWEB)

    Romanov, G.A.; Kulaeva, O.N.; Taryan, V.Y.

    1986-01-01

    Highly labelled tritium-zeatin was used in the work to clarify for the first time a protein factor that is present in cytokinin-sensitive vegetative organs of plants (barley leaves) and which possesses the properties of a cytokinin receptor. Aliquots of tritium-zeatin were mixed with a solution of protein and incubated for several hours in buffer. Following incubation, protein was precipitated by ammonium sulfate at 90% of saturation, and radioactivity of the precipitate was checked in a dioxane scintillator with an efficiency of about 35%. It is shown that the characteristics of interaction of the clarified specific protein sites with cytokinins in regard to a number of criteria correspond to the characteristics expected of receptors of these phytohormones.

  12. Characterization of heme-binding properties of Paracoccus denitrificans Surf1 proteins.

    Science.gov (United States)

    Hannappel, Achim; Bundschuh, Freya A; Ludwig, Bernd

    2011-05-01

    Biogenesis of cytochrome c oxidase (COX) is a highly complex process involving >30 chaperones in eukaryotes; those required for the incorporation of the copper and heme cofactors are also conserved in bacteria. Surf1, associated with heme a insertion and with Leigh syndrome if defective in humans, is present as two homologs in the soil bacterium Paracoccus denitrificans, Surf1c and Surf1q. In an in vitro interaction assay, the heme a transfer from purified heme a synthase, CtaA, to Surf1c was followed, and both Surf proteins were tested for their heme a binding properties. Mutation of four strictly conserved amino acid residues within the transmembrane part of each Surf1 protein confirmed their requirement for heme binding. Interestingly the mutation of a tryptophan residue in transmembrane helix II (W200 in Surf1c and W209 in Surf1q) led to a drastic switch in the heme composition, with Surf1 now being populated mostly by heme o, the intermediate in the heme a biosynthetic pathway. This tryptophan residue discriminates between the two heme moieties, apparently coordinates the formyl group of heme a, and most likely presents the cofactor in a spatial orientation suitable for optimal transfer to its target site within subunit I of cytochrome c oxidase. PMID:21418525

  13. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  14. Theoretical studies of binding of mannose-binding protein to monosaccharides

    Science.gov (United States)

    Aida-Hyugaji, Sachiko; Takano, Keiko; Takada, Toshikazu; Hosoya, Haruo; Kojima, Naoya; Mizuochi, Tsuguo; Inoue, Yasushi

    2004-11-01

    Binding properties of mannose-binding protein (MBP) to monosaccharides are discussed based on ab initio molecular orbital calculations for cluster models constructed. The calculated binding energies indicate that MBP has an affinity for N-acetyl- D-glucosamine, D-mannose, L-fucose, and D-glucose rather than D-galactose and N-acetyl- D-galactosamine, which is consistent with the biochemical experimental results. Electrostatic potential surfaces at the binding site of four monosaccharides having binding properties matched well with that of MBP. A vacant frontier orbital was found to be localized around the binding site of MBP, suggesting that MBP-monosaccharide interaction may occur through electrostatic and orbital interactions.

  15. Cholesterol-binding viral proteins in virus entry and morphogenesis.

    Science.gov (United States)

    Schroeder, Cornelia

    2010-01-01

    Up to now less than a handful of viral cholesterol-binding proteins have been characterized, in HIV, influenza virus and Semliki Forest virus. These are proteins with roles in virus entry or morphogenesis. In the case of the HIV fusion protein gp41 cholesterol binding is attributed to a cholesterol recognition consensus (CRAC) motif in a flexible domain of the ectodomain preceding the trans-membrane segment. This specific CRAC sequence mediates gp41 binding to a cholesterol affinity column. Mutations in this motif arrest virus fusion at the hemifusion stage and modify the ability of the isolated CRAC peptide to induce segregation of cholesterol in artificial membranes.Influenza A virus M2 protein co-purifies with cholesterol. Its proton translocation activity, responsible for virus uncoating, is not cholesterol-dependent, and the transmembrane channel appears too short for integral raft insertion. Cholesterol binding may be mediated by CRAC motifs in the flexible post-TM domain, which harbours three determinants of binding to membrane rafts. Mutation of the CRAC motif of the WSN strain attenuates virulence for mice. Its affinity to the raft-non-raft interface is predicted to target M2 protein to the periphery of lipid raft microdomains, the sites of virus assembly. Its influence on the morphology of budding virus implicates M2 as factor in virus fission at the raft boundary. Moreover, M2 is an essential factor in sorting the segmented genome into virus particles, indicating that M2 also has a role in priming the outgrowth of virus buds.SFV E1 protein is the first viral type-II fusion protein demonstrated to directly bind cholesterol when the fusion peptide loop locks into the target membrane. Cholesterol binding is modulated by another, proximal loop, which is also important during virus budding and as a host range determinant, as shown by mutational studies. PMID:20213541

  16. Detergent activation of the binding protein in the folate radioassay

    International Nuclear Information System (INIS)

    A minor cow's whey protein associated with β-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to β-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants

  17. MG-132 inhibits the TCDD-mediated induction of Cyp1a1 at the catalytic activity but not the mRNA or protein levels in Hepa 1c1c7 cells.

    Science.gov (United States)

    Anwar-Mohamed, Anwar; Elbekai, Reem H; El-Kadi, Ayman O S

    2008-11-10

    Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced degradation of aryl hydrocarbon receptor (AhR) is inhibited by MG-132, a potent inhibitor of the 26S proteasome. Therefore, the current study aims to address the effect of MG-132 on the AhR-regulated gene, cytochrome P450 1a1 (Cyp1a1), using murine hepatoma Hepa 1c1c7 cells. Our results showed that MG-132 at the highest concentration tested, 10 microM significantly increased the Cyp1a1 at mRNA, protein and catalytic activity levels through a transcriptional mechanism. On the other hand, MG-132 further potentiated the TCDD-mediated induction of Cyp1a1 at mRNA but not at protein level. In contrast, MG-132 significantly inhibited the TCDD-mediated induction of Cyp1a1 catalytic activity. In addition, we showed that the decrease in Cyp1a1 catalytic activity is not Cyp specific, as MG-132 significantly inhibited Cyp2b1 and total cytochrome P450 catalytic activities. These results prompted us to examine the effect of MG-132 on total cellular heme content and heme oxygenase-1 (HO-1) mRNA, a rate limiting enzyme of heme degradation. Our results showed that MG-132 significantly induced HO-1 mRNA in a concentration-dependent manner. Furthermore, MG-132 potentiated the induction of HO-1 mRNA by TCDD in a concentration-dependent manner. The induction of HO-1 mRNA level coincided with a decrease in total cellular heme content. In conclusion, the present study demonstrates for the first time that MG-132, despite of increasing Cyp1a1 mRNA expression, it decreases its activity probably through decreasing its heme content. PMID:18835339

  18. Drug Promiscuity in PDB: Protein Binding Site Similarity Is Key.

    Directory of Open Access Journals (Sweden)

    V Joachim Haupt

    Full Text Available Drug repositioning applies established drugs to new disease indications with increasing success. A pre-requisite for drug repurposing is drug promiscuity (polypharmacology - a drug's ability to bind to several targets. There is a long standing debate on the reasons for drug promiscuity. Based on large compound screens, hydrophobicity and molecular weight have been suggested as key reasons. However, the results are sometimes contradictory and leave space for further analysis. Protein structures offer a structural dimension to explain promiscuity: Can a drug bind multiple targets because the drug is flexible or because the targets are structurally similar or even share similar binding sites? We present a systematic study of drug promiscuity based on structural data of PDB target proteins with a set of 164 promiscuous drugs. We show that there is no correlation between the degree of promiscuity and ligand properties such as hydrophobicity or molecular weight but a weak correlation to conformational flexibility. However, we do find a correlation between promiscuity and structural similarity as well as binding site similarity of protein targets. In particular, 71% of the drugs have at least two targets with similar binding sites. In order to overcome issues in detection of remotely similar binding sites, we employed a score for binding site similarity: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary structural binding site alignments. Three representative examples, namely the anti-cancer drug methotrexate, the natural product quercetin and the anti-diabetic drug acarbose are discussed in detail. Our findings suggest that global structural and binding site similarity play a more important role to explain the observed drug promiscuity in the PDB than physicochemical drug properties like hydrophobicity or molecular weight. Additionally, we find ligand

  19. Protein-protein binding affinities calculated using the LIE method

    OpenAIRE

    Andberg, Tor Arne Heim

    2011-01-01

    Absolute binding free energies for the third domain of the turkey ovomucoid inhibitor in complex with Streptomyces griseus proteinase B and porcine pancreatic elastase has been calculated using the linear interaction energy method.

  20. The Role Stress Granules and RNA Binding Proteins in Neurodegeneration

    OpenAIRE

    Vanderweyde, Tara; Youmans, Katie; Liu-Yesucevitz, Liqun; Wolozin, Benjamin

    2013-01-01

    The eukaryotic stress response involves translational suppression of non-housekeeping proteins and the sequestration of unnecessary mRNA transcripts into stress granules (SGs). This process is dependent on mRNA binding proteins (RBPs) that interact with capped mRNA transcripts through RNA recognition motifs, and exhibit reversible aggregation through hydrophobic poly-glycine domains, some of which are homologous to yeast prion proteins. The activity and aggregation of RBPs appears to be impor...

  1. Pentatricopeptide repeats: Modular blocks for building RNA-binding proteins

    OpenAIRE

    Filipovska, Aleksandra; Rackham, Oliver

    2013-01-01

    Pentatricopeptide repeat (PPR) proteins control diverse aspects of RNA metabolism across the eukaryotic domain. Recent computational and structural studies have provided new insights into how they recognize RNA, and show that the recognition is sequence-specific and modular. The modular code for RNA-binding by PPR proteins holds great promise for the engineering of new tools to target RNA and identifying RNAs bound by natural PPR proteins.

  2. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    OpenAIRE

    Villamor, J. G.; Kaschani, F.; Colby, T; Oeljeklaus, J.; Zhao, D; Kaiser, M.; Patricelli, M. P.; R. A. L. van der Hoorn

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding prot...

  3. A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain

    Science.gov (United States)

    Wang, W.; Takezawa, D.; Narasimhulu, S. B.; Reddy, A. S. N.; Poovaiah, B. W.

    1996-01-01

    Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

  4. Natural history of S-adenosylmethionine-binding proteins

    Directory of Open Access Journals (Sweden)

    Mushegian Arcady R

    2005-10-01

    Full Text Available Abstract Background S-adenosylmethionine is a source of diverse chemical groups used in biosynthesis and modification of virtually every class of biomolecules. The most notable reaction requiring S-adenosylmethionine, transfer of methyl group, is performed by a large class of enzymes, S-adenosylmethionine-dependent methyltransferases, which have been the focus of considerable structure-function studies. Evolutionary trajectories of these enzymes, and especially of other classes of S-adenosylmethionine-binding proteins, nevertheless, remain poorly understood. We addressed this issue by computational comparison of sequences and structures of various S-adenosylmethionine-binding proteins. Results Two widespread folds, Rossmann fold and TIM barrel, have been repeatedly used in evolution for diverse types of S-adenosylmethionine conversion. There were also cases of recruitment of other relatively common folds for S-adenosylmethionine binding. Several classes of proteins have unique unrelated folds, specialized for just one type of chemistry and unified by the theme of internal domain duplications. In several cases, functional divergence is evident, when evolutionarily related enzymes have changed the mode of binding and the type of chemical transformation of S-adenosylmethionine. There are also instances of functional convergence, when biochemically similar processes are performed by drastically different classes of S-adenosylmethionine-binding proteins. Comparison of remote sequence similarities and analysis of phyletic patterns suggests that the last universal common ancestor of cellular life had between 10 and 20 S-adenosylmethionine-binding proteins from at least 5 fold classes, providing for S-adenosylmethionine formation, polyamine biosynthesis, and methylation of several substrates, including nucleic acids and peptide chain release factor. Conclusion We have observed several novel relationships between families that were not known to be

  5. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  6. A Genetic Screen Identifies Putative Targets and Binding Partners of CREB-Binding Protein in the Developing Drosophila Eye

    OpenAIRE

    Anderson, Jason; Bhandari, Rohan; Kumar, Justin P.

    2005-01-01

    Drosophila CREB-binding protein (dCBP) is a very large multidomain protein, which belongs to the CBP/p300 family of proteins that were first identified by their ability to bind the CREB transcription factor and the adenoviral protein E1. Since then CBP has been shown to bind to >100 additional proteins and functions in a multitude of different developmental contexts. Among other activities, CBP is known to influence development by remodeling chromatin, by serving as a transcriptional coactiva...

  7. Tetrapyrrole binding affinity of the murine and human p22HBP heme-binding proteins.

    Science.gov (United States)

    Micaelo, Nuno M; Macedo, Anjos L; Goodfellow, Brian J; Félix, Vítor

    2010-11-01

    We present the first systematic molecular modeling study of the binding properties of murine (mHBP) and human (hHBP) p22HBP protein (heme-binding protein) with four tetrapyrrole ring systems belonging to the heme biosynthetic pathway: iron protoporphyrin IX (HEMIN), protoporphyrin IX (PPIX), coproporphyrin III (CPIII), coproporphyrin I (CPI). The relative binding affinities predicted by our computational study were found to be similar to those observed experimentally, providing a first rational structural analysis of the molecular recognition mechanism, by p22HBP, toward a number of different tetrapyrrole ligands. To probe the structure of these p22HBP protein complexes, docking, molecular dynamics and MM-PBSA methodologies supported by experimental NMR ring current shift data have been employed. The tetrapyrroles studied were found to bind murine p22HBP with the following binding affinity order: HEMIN> PPIX> CPIII> CPI, which ranged from -22.2 to -6.1 kcal/mol. In general, the protein-tetrapyrrole complexes are stabilized by non-bonded interactions between the tetrapyrrole propionate groups and basic residues of the protein, and by the preferential solvation of the complex compared to the unbound components. PMID:20800521

  8. Sub-cellular localisation of fukutin related protein in different cell lines and in the muscle of patients with MDC1C and LGMD2I

    DEFF Research Database (Denmark)

    Torelli, Silvia; Brown, Susan C; Brockington, Martin; Dolatshad, Nazanin F; Jimenez, Cecilia; Skordis, Leigh; Feng, Lucy H; Merlini, Luciano; Jones, David Hilton; Romero, Norma; Wewer, Ulla; Voit, Thomas; Sewry, Caroline A; Noguchi, Satoru; Nishino, Ichizo; Muntoni, Francesco

    2005-01-01

    MDC1C and LGMD2I are two allelic forms of muscular dystrophies caused by mutations in the gene encoding for fukutin related protein (FKRP). FKRP encodes for a putative glycosyltransferase, the precise function of which is unknown. However, the marked reduction of alpha-dystroglycan glycosylation in...... myotubes and in transverse sections of normal skeletal and cardiac muscle, endogenous FKRP surrounded the myonuclei. This localisation was unaffected in the skeletal muscle of patients with MDC1C and LGMD2I carrying various FKRP mutations. These observations imply a specific role for FKRP during striated...

  9. Structure-Function Analysis of the Anopheles gambiae LRIM1/APL1C Complex and its Interaction with Complement C3-Like Protein TEP1

    OpenAIRE

    Povelones, Michael; Upton, Leanna M.; Sala, Katarzyna A.; Christophides, George K.

    2011-01-01

    Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-b...

  10. Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1.

    OpenAIRE

    Michael Povelones; Upton, Leanna M.; Sala, Katarzyna A.; Christophides, George K.

    2011-01-01

    Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-b...

  11. Predicting the binding patterns of hub proteins: a study using yeast protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Carson M Andorf

    Full Text Available BACKGROUND: Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH with one or two binding sites, or multiple-interface hubs (MIH with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations or party hubs (i.e., simultaneously interact with multiple partners. METHODOLOGY: Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques. CONCLUSIONS: Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions. AVAILABILITY: We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu.

  12. Characterization of adenosine binding proteins in human placental membranes

    International Nuclear Information System (INIS)

    We have characterized two adenosine binding proteins in human placenta. In membranes, one site is detected with [3H] -N-ethylcarboxamidoadenosine ([3H]NECA). This site is similar to the adenosine A2 receptor. We call this site the adenosine A2-like binding site. In detergent extracts, the second site is detected and has the characteristics of an adenosine A1 receptor. The soluble adenosine A2-like binding site cannot be detected without a rapid assay. Binding to the adenosine A1 receptor with [3H]-2-chloroadenosine and [3H]NECA is time dependent, saturable, and reversible. Equilibrium displacement analysis with adenosine agonists reveals an A1 specificity: 2-chloroadenosine > R-phenylisopropyladenosine > 5'-N-ethylcarboxamidoadenosine. The antagonist potency order is 1,3-diethyl-8-phenylxanthine > isobutylmethylxanthine > theophylline. Competition analysis of membranes with the A,-selective ligands [3H]-cyclohexyladenosine [3H] cylopentylxanthine revealed adenosine A1 agonist and antagonist potency orders. We have purified the adenosine A2-like binding site. The adenosine A2-like binding site is an ubiquitous major cellular protein. It is glycosylated, highly asymmetric, and acidic. The native protein is an homodimer with a subunit molecular mass of 98 kDa. The sedimentation coefficient and partial specific volume of the binding complex are 6.9 s and 0.698 ml/g, respectively. The Stokes' radius is 70 Angstrom. The native molecular mass of the detergent-protein complex is 230 kDa. The adenosine A2-like binding site has an agonist potency order of 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine >> R-phenylisopropyladenosine and an antagonist potency order of isobutylmethylxanthine > theophylline >> 1,3-diethyl-8-phenylxanthine

  13. Characterization of adenosine binding proteins in human placental membranes

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, K.A.

    1989-01-01

    We have characterized two adenosine binding proteins in human placenta. In membranes, one site is detected with ({sup 3}H) -N-ethylcarboxamidoadenosine (({sup 3}H)NECA). This site is similar to the adenosine A{sub 2} receptor. We call this site the adenosine A{sub 2}-like binding site. In detergent extracts, the second site is detected and has the characteristics of an adenosine A{sub 1} receptor. The soluble adenosine A{sub 2}-like binding site cannot be detected without a rapid assay. Binding to the adenosine A{sub 1} receptor with ({sup 3}H)-2-chloroadenosine and ({sup 3}H)NECA is time dependent, saturable, and reversible. Equilibrium displacement analysis with adenosine agonists reveals an A{sub 1} specificity: 2-chloroadenosine > R-phenylisopropyladenosine > 5{prime}-N-ethylcarboxamidoadenosine. The antagonist potency order is 1,3-diethyl-8-phenylxanthine > isobutylmethylxanthine > theophylline. Competition analysis of membranes with the A,-selective ligands ({sup 3}H)-cyclohexyladenosine ({sup 3}H) cylopentylxanthine revealed adenosine A{sub 1} agonist and antagonist potency orders. We have purified the adenosine A{sub 2}-like binding site. The adenosine A{sub 2}-like binding site is an ubiquitous major cellular protein. It is glycosylated, highly asymmetric, and acidic. The native protein is an homodimer with a subunit molecular mass of 98 kDa. The sedimentation coefficient and partial specific volume of the binding complex are 6.9 s and 0.698 ml/g, respectively. The Stokes' radius is 70 {Angstrom}. The native molecular mass of the detergent-protein complex is 230 kDa. The adenosine A{sub 2}-like binding site has an agonist potency order of 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine >> R-phenylisopropyladenosine and an antagonist potency order of isobutylmethylxanthine > theophylline >> 1,3-diethyl-8-phenylxanthine.

  14. Identification of albumin-binding proteins in capillary endothelial cells

    OpenAIRE

    1988-01-01

    Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of...

  15. Yeast TATA-binding protein TFIID binds to TATA elements with both consensus and nonconsensus DNA sequences.

    OpenAIRE

    S. Hahn; Buratowski, S.; Sharp, P A; Guarente, L

    1989-01-01

    The DNA binding properties of the yeast TATA element-binding protein TFIID were investigated. The affinity (apparent equilibrium dissociation constant) of TFIID for the adenovirus major late promoter consensus TATA element is 2 x 10(-9) M, a value similar to the affinity of gene-specific regulatory proteins for their binding sites. TFIID binding is highly specific and recognizes nonspecific sites with approximately 10(5)-fold lower affinity. Despite this specificity, TFIID also binds with hig...

  16. Differential plasma protein binding to metal oxide nanoparticles

    International Nuclear Information System (INIS)

    Nanoparticles rapidly interact with the proteins present in biological fluids, such as blood. The proteins that are adsorbed onto the surface potentially dictate the biokinetics of the nanomaterials and their fate in vivo. Using nanoparticles with different sizes and surface characteristics, studies have reported the effects of physicochemical properties on the composition of adsorbed plasma proteins. However, to date, few studies have been conducted focusing on the nanoparticles that are commonly exposed to the general public, such as the metal oxides. Using previously established ultracentrifugation approaches, two-dimensional gel electrophoresis and mass spectrometry, the current study investigated the binding of human plasma proteins to commercially available titanium dioxide, silicon dioxide and zinc oxide nanoparticles. We found that, despite these particles having similar surface charges in buffer, they bound different plasma proteins. For TiO2, the shape of the nanoparticles was also an important determinant of protein binding. Agglomeration in water was observed for all of the nanoparticles and both TiO2 and ZnO further agglomerated in biological media. This led to an increase in the amount and number of different proteins bound to these nanoparticles. Proteins with important biological functions were identified, including immunoglobulins, lipoproteins, acute-phase proteins and proteins involved in complement pathways and coagulation. These results provide important insights into which human plasma proteins bind to particular metal oxide nanoparticles. Because protein absorption to nanoparticles may determine their interaction with cells and tissues in vivo, understanding how and why plasma proteins are adsorbed to these particles may be important for understanding their biological responses.

  17. The liver fatty acid binding protein--comparison of cavity properties of intracellular lipid-binding proteins.

    Science.gov (United States)

    Thompson, J; Ory, J; Reese-Wagoner, A; Banaszak, L

    1999-02-01

    The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common beta-barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited 'portal' region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound. PMID:10331654

  18. Binding dynamics of single-stranded DNA binding proteins to fluctuating bubbles in breathing DNA

    International Nuclear Information System (INIS)

    We investigate the dynamics of a single local denaturation zone in a DNA molecule, a so-called DNA bubble, in the presence of single-stranded DNA binding proteins (SSBs). In particular, we develop a dynamical description of the process in terms of a two-dimensional master equation for the time evolution of the probability distribution of having a bubble of size m with n bound SSBs, for the case when m and n are the slowest variables in the system. We derive explicit expressions for the equilibrium statistical weights for a given m and n, which depend on the statistical weight u associated with breaking a base-pair interaction, the loop closure exponent c, the cooperativity parameter σ0, the SSB size λ, and binding strength κ. These statistical weights determine, through the detailed balance condition, the transfer coefficient in the master equation. For the case of slow and fast binding dynamics the problem can be reduced to one-dimensional master equations. In the latter case, we perform explicitly the adiabatic elimination of the fast variable n. Furthermore, we find that for the case that the loop closure is neglected and the binding dynamics is vanishing (but with arbitrary σ0) the eigenvalues and the eigenvectors of the master equation can be obtained analytically, using an orthogonal polynomial approach. We solve the general case numerically (i.e., including SSB binding and the loop closure) as a function of statistical weight u, binding protein size λ, and binding strength κ, and compare to the fast and slow binding limits. In particular, we find that the presence of SSBs in general increases the relaxation time, compared to the case when no binding proteins are present. By tuning the parameters, we can drive the system from regular bubble fluctuation in the absence of SSBs to full denaturation, reflecting experimental and in vivo situations

  19. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  20. The plasma protein binding of HIDA

    International Nuclear Information System (INIS)

    By using Sephadex gel column chromatography to separate substances into their various components according to molecular weight, we have investigated the effect of incubating several brands of HIDA in plasma, in vitro. The results show that such incubation has no effect on either dimethyl HIDA, or diethyl HIDA, but that in the case of para-butyl HIDA, incubation in plasma increases the Rf value to that of HSA (human serum albumin). This indicates that para-butyl HIDA becomes bound to plasma proteins, in contrast to both dimethyl HIDA and diethyl HIDA. (orig.)

  1. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

    Science.gov (United States)

    Sharifi, Reza; Morra, Rosa; Denise Appel, C; Tallis, Michael; Chioza, Barry; Jankevicius, Gytis; Simpson, Michael A; Matic, Ivan; Ozkan, Ege; Golia, Barbara; Schellenberg, Matthew J; Weston, Ria; Williams, Jason G; Rossi, Marianna N; Galehdari, Hamid; Krahn, Juno; Wan, Alexander; Trembath, Richard C; Crosby, Andrew H; Ahel, Dragana; Hay, Ron; Ladurner, Andreas G; Timinszky, Gyula; Williams, R Scott; Ahel, Ivan

    2013-01-01

    Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans. PMID:23481255

  2. Characterization of a calmodulin binding protein kinase from Arabidopsis thalian

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A full-length calmodulin binding protein kinase cDNA, AtCBK1, from Arabidopsis has been isolated by screening of an Arabidopsis cDNA library and by 5′-RACE. Northern blot and in situ hybridization indicated that the expression of AtCBK1 was more abundant in the vascular bundles and the meristems than in other tissues. The phylogenetic analyses reveal that AtCBK1 is different from animal CaMKs and it falls into CRK subgroup, indicating that they may come from different ancestors. The result suggests that AtCBK1 encodes a CaM-binding serine/threonine protein kinase.

  3. Detection of Fibronectin-Binding Proteins in Clostridium perfringens.

    Directory of Open Access Journals (Sweden)

    Yokoyama,Masako

    2006-12-01

    Full Text Available Clostridium perfringens is an anaerobic spore-forming pathogen of humans and animals. C. perfringens type A strains, 13, CPN50, and NCTC8237, isolated from human gas gangrene, bound specifically to human fi bronectin (Fn. The trypsin-treatment of the bacterial cells significantly reduced the Fn-binding. A ligand blotting analysis of all three C. perfringens strains revealed that 5 protein bands of 34 kDa, 29 kDa, 26 kDa, 17 kDa, and 12 kDa specifically bound to biotinylated Fn. These results suggest that C. perfringens possesses certain Fn-binding proteins on the cell surface.

  4. The gene encoding acyl-CoA-binding protein is subject to metabolic regulation by both sterol regulatory element-binding protein and peroxisome proliferator-activated receptor alpha in hepatocytes

    DEFF Research Database (Denmark)

    Sandberg, Maria B; Bloksgaard, Maria; Duran-Sandoval, Daniel; Duval, Caroline; Staels, Bart; Mandrup, Susanne

    2005-01-01

    The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular lipid-binding protein that transports acylCoA esters. The protein is expressed in most cell types at low levels; however, expression is particularly high in cells with a high turnover of fatty acids. Here we confirm a previous...... observation that ACBP expression in rodent liver is down-regulated by fasting, and we show that insulin but not glucose is the inducer of ACBP expression in primary rat hepatocytes. In keeping with the regulation by insulin, we show that ACBP is a sterol regulatory element-binding protein 1c (SREBP-1c) target...... that ACBP expression is significantly lower in livers from PPARalpha knock-out mice than in livers from wild type mice. In conclusion, expression of ACBP in rodent hepatocytes is subject to dual metabolic regulation by PPARalpha and SREBP-1c, which may reflect the need for ACBP during lipogenic as well...

  5. Binding-regulated click ligation for selective detection of proteins.

    Science.gov (United States)

    Cao, Ya; Han, Peng; Wang, Zhuxin; Chen, Weiwei; Shu, Yongqian; Xiang, Yang

    2016-04-15

    Herein, a binding-regulated click ligation (BRCL) strategy for endowing selective detection of proteins is developed with the incorporation of small-molecule ligand and clickable DNA probes. The fundamental principle underlying the strategy is the regulating capability of specific protein-ligand binding against the ligation between clickable DNA probes, which could efficiently combine the detection of particular protein with enormous DNA-based sensing technologies. In this work, the feasibly of the BRCL strategy is first verified through agarose gel electrophoresis and electrochemical impedance spectroscopy measurements, and then confirmed by transferring it to a nanomaterial-assisted fluorescence assay. Significantly, the BRCL strategy-based assay is able to respond to target protein with desirable selectivity, attributing to the specific recognition between small-molecule ligand and its target. Further experiments validate the general applicability of the sensing method by tailoring the ligand toward different proteins (i.e., avidin and folate receptor), and demonstrate its usability in complex biological samples. To our knowledge, this work pioneers the practice of click chemistry in probing specific small-molecule ligand-protein binding, and therefore may pave a new way for selective detection of proteins. PMID:26599478

  6. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

  7. All-Purpose Containers? Lipid-Binding Protein - Drug Interactions.

    Directory of Open Access Journals (Sweden)

    Tiziana Beringhelli

    Full Text Available The combined use of in vitro (19F-NMR and in silico (molecular docking procedures demonstrates the affinity of a number of human calycins (lipid-binding proteins from ileum, liver, heart, adipose tissue and epidermis, and retinol-binding protein from intestine for different drugs (mainly steroids and vastatins. Comparative evaluations on the complexes outline some of the features relevant for interaction (non-polar character of the drugs; amino acids and water molecules in the protein calyx most often involved in binding. Dissociation constants (Ki for drugs typically lie in the same range as Ki for natural ligands; in most instances (different proteins and docking conditions, vastatins are the strongest interactors, with atorvastatin ranking top in half of the cases. The affinity of some calycins for some of the vastatins is in the order of magnitude of the drug Cmax after systemic administration in humans. The possible biological implications of this feature are discussed in connection with drug delivery parameters (route of administration, binding to carrier proteins, distribution to, and accumulation in, human tissues.

  8. Drug-drug plasma protein binding interactions of ivacaftor.

    Science.gov (United States)

    Schneider, Elena K; Huang, Johnny X; Carbone, Vincenzo; Baker, Mark; Azad, Mohammad A K; Cooper, Matthew A; Li, Jian; Velkov, Tony

    2015-06-01

    Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR-protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co-administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co-administered CF drugs for human serum albumin (HSA) and α1 -acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site-selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug-drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug-drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug-drug interactions of ivacaftor with co-administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. PMID:25707701

  9. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

    OpenAIRE

    Sharifi, Reza; Morra, Rosa; Denise Appel, C; Tallis, Michael; Chioza, Barry; Jankevicius, Gytis; Simpson, Michael A.; Matic, Ivan; Ozkan, Ege; Golia, Barbara; Schellenberg, Matthew J.; Weston, Ria; Williams, Jason G.; Rossi, Marianna N.; Galehdari, Hamid

    2013-01-01

    Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase...

  10. Fatty Acid- and Retinoid-binding Proteins Have Distinct Binding Pockets for the Two Types of Cargo*

    OpenAIRE

    Jordanova, Rositsa; Groves, Matthew R.; Kostova, Elena; Woltersdorf, Christian; Liebau, Eva; Tucker, Paul A.

    2009-01-01

    Parasitic nematodes cause serious diseases in humans, animals, and plants. They have limited lipid metabolism and are reliant on lipid-binding proteins to acquire these metabolites from their hosts. Several structurally novel families of lipid-binding proteins in nematodes have been described, including the fatty acid- and retinoid-binding protein family (FAR). In Caenorhabditis elegans, used as a model for studying parasitic nematodes, eight C. elegans FAR proteins have been described. The c...

  11. Surf1, associated with Leigh syndrome in humans, is a heme-binding protein in bacterial oxidase biogenesis.

    Science.gov (United States)

    Bundschuh, Freya A; Hannappel, Achim; Anderka, Oliver; Ludwig, Bernd

    2009-09-18

    Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool. PMID:19625251

  12. Drug bioactivation, covalent binding to target proteins and toxicity relevance.

    Science.gov (United States)

    Zhou, Shufeng; Chan, Eli; Duan, Wei; Huang, Min; Chen, Yu-Zong

    2005-01-01

    A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients

  13. Plant RNA binding proteins for control of RNA virus infection

    OpenAIRE

    Huh, Sung Un; Paek, Kyung-Hee

    2013-01-01

    Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific b...

  14. Free enthalpies of replacing water molecules in protein binding pockets.

    Science.gov (United States)

    Riniker, Sereina; Barandun, Luzi J; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design. PMID:23247390

  15. The distribution of ligand-binding pockets around protein-protein interfaces suggests a general mechanism for pocket formation

    OpenAIRE

    Gao, Mu; Skolnick, Jeffrey

    2012-01-01

    Protein-protein and protein-ligand interactions are ubiquitous in a biological cell. Here, we report a comprehensive study of the distribution of protein-ligand interaction sites, namely ligand-binding pockets, around protein-protein interfaces where protein-protein interactions occur. We inspected a representative set of 1,611 representative protein-protein complexes and identified pockets with a potential for binding small molecule ligands. The majority of these pockets are within a 6 Å dis...

  16. Pumilio Puf domain RNA-binding proteins in Arabidopsis.

    Science.gov (United States)

    Abbasi, Nazia; Park, Youn-Il; Choi, Sang-Bong

    2011-03-01

    Pumilio proteins are a class of RNA-binding proteins harboring Puf domains (or PUM-HD; Pumilio-Homology Domain), named after the founding members, Pumilio (from Drosophila melanogaster) and FBF (Fem-3 mRNA-Binding Factor from Caenorhabditis elegans). The domains contain multiple tandem repeats each of which recognizes one RNA base and is comprised of 35-39 amino acids. Puf domain proteins have been reported in organisms ranging from single-celled yeast to higher multicellular eukaryotes, such as humans and plants. In yeast and animals, they are involved in a variety of posttranscriptional RNA metabolism including RNA decay, RNA transport, rRNA processing and translational repression. However, their roles in plants are largely unknown. Recently, we have characterized the first member of the Puf family of RNA-binding proteins, APUM23, in Arabidopsis. Here, we discuss and summarize the diverse roles and targets of Puf proteins previously reported in other organisms and then highlight the potential regulatory roles of Puf proteins in Arabidopsis, using our recent study as an example. PMID:21350339

  17. Calcium-binding ability of soy protein hydrolysates

    Institute of Scientific and Technical Information of China (English)

    Xiao Lan Bao; Mei Song; Jing Zhang; Yang Chen; Shun Tang Guo

    2007-01-01

    This present study investigated the ability of various soy protein hydrolysates (SPHs) in binding calcium. It was demonstrated that the amount of Ca-bound depended greatly on the SPHs obtained using different proteases, which included: neutrase,flavourzyme, protease M and pepsin. The maximum level of Ca-bound (66.9 mg/g) occurred when protease M was used to hydrolyze soy protein. Peptide fragments exhibiting high Ca-binding capacity had molecular weights of either 14.4 or 8-9 kDa. The level of Ca-bound increased linearly with the increment of carboxyl content in SPHs, and further deamidation on SPHs from protease M improved Ca-binding of the hydrolysate.

  18. Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

    Science.gov (United States)

    Rowland, Andrew; Hallifax, David; Nussio, Matthew R; Shapter, Joseph G; Mackenzie, Peter I; Brian Houston, J; Knights, Kathleen M; Miners, John O

    2015-01-01

    1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP. PMID:25801059

  19. Identification of Enhancer Binding Proteins Important for Myxococcus xanthus Development▿

    OpenAIRE

    Giglio, Krista M.; Eisenstatt, Jessica; Garza, Anthony G.

    2009-01-01

    Enhancer binding proteins (EBPs) control the temporal expression of fruiting body development-associated genes in Myxococcus xanthus. Eleven previously uncharacterized EBP genes were inactivated. Six EBP gene mutations produced minor but reproducible defects in fruiting body development. One EBP gene mutation that affected A-motility produced strong developmental defects.

  20. The Role of Microtubule End Binding (EB) Proteins in Ciliogenesis

    DEFF Research Database (Denmark)

    Schrøder, Jacob Morville

    biflagellate green alga Chlamydomonas (Pedersen et al., 2003), and is required for ciliogenesis in mouse fibroblasts (Schroder et al., 2007). However, the exact mechanism(s) involved and roles of the two additional mammalian members of the end binding (EB) protein family, EB2 and EB3, in ciliogenesis are...

  1. Monomeric Yeast Frataxin is an Iron-Binding Protein

    Energy Technology Data Exchange (ETDEWEB)

    Cook,J.; Bencze, K.; Jankovic, A.; Crater, A.; Busch, C.; Bradley, P.; Stemmler, A.; Spaller, M.; Stemmler, T.

    2006-01-01

    Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.

  2. Monomeric Yeast Frataxin is an Iron-Binding Protein

    International Nuclear Information System (INIS)

    Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron

  3. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation

    OpenAIRE

    Weidmann, Chase A.; Raynard, Nathan A.; Blewett, Nathan H.; Van Etten, Jamie; Goldstrohm, Aaron C.

    2014-01-01

    This article analyzes the mechanism by which Pumilio represses the translation of its targets. The results show, rather surprisingly, that promotion of deadenylation is not required for expression. Instead, Pumilio interacts with poly(A) binding protein and somehow interferes with its activity.

  4. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

    DEFF Research Database (Denmark)

    Jensen, P Y; Bonander, N; Møller, L B;

    1999-01-01

    We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spec...

  5. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation.

    Science.gov (United States)

    Weidmann, Chase A; Raynard, Nathan A; Blewett, Nathan H; Van Etten, Jamie; Goldstrohm, Aaron C

    2014-08-01

    PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3' untranslated regions of target mRNAs. In this study, we investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. Here, we evaluated a previously proposed model wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, we found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, we determined that the poly(A) is necessary for repression by the RBD. Our results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, we show that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, our data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation. PMID:24942623

  6. In vivo threonine phosphorylation of immunoglobulin binding protein (BiP) maps to its protein binding domain

    OpenAIRE

    Gaut, James R.

    1997-01-01

    lmmunoglobin binding protein (BiP) molecules exist as both monomers and oligomers and phosphorylated BiP is restricted to the oligomeric pool. Modified BiP is not bound to proteins such as immunoglobulin heavy chain and consequently, may constitute an inactive form. Unlike earlier analysis of mammalian BiP isolated by two-dimensional gel electrophoresis, results here demonstrated that immunoprecipitated BiP displayed predominantly threonine phosphorylation with only a trace of detectable phos...

  7. Engineering periplasmic ligand binding proteins as glucose nanosensors

    Directory of Open Access Journals (Sweden)

    Constance J. Jeffery

    2011-01-01

    Full Text Available Diabetes affects over 100 million people worldwide. Better methods for monitoring blood glucose levels are needed for improving disease management. Several labs have previously made glucose nanosensors by modifying members of the periplasmic ligand binding protein superfamily. This minireview summarizes recent developments in constructing new versions of these proteins that are responsive within the physiological range of blood glucose levels, employ new reporter groups, and/or are more robust. These experiments are important steps in the development of novel proteins that have the characteristics needed for an implantable glucose nanosensor for diabetes management: specificity for glucose, rapid response, sensitivity within the physiological range of glucose concentrations, reproducibility, and robustness.

  8. Polyunsaturated Fatty Acids Selectively Suppress Sterol Regulatory Element-binding Protein-1 through Proteolytic Processing and Autoloop Regulatory Circuit*

    OpenAIRE

    Takeuchi, Yoshinori; Yahagi, Naoya; Izumida, Yoshihiko; Nishi, Makiko; Kubota, Midori; Teraoka, Yuji; Yamamoto, Takashi; Matsuzaka, Takashi; Nakagawa, Yoshimi; Sekiya, Motohiro; Iizuka, Yoko; Ohashi, Ken; Osuga, Jun-ichi; Gotoda, Takanari; Ishibashi, Shun

    2010-01-01

    Sterol regulatory element-binding protein (SREBP)-1 is a key transcription factor for the regulation of lipogenic enzyme genes in the liver. Polyunsaturated fatty acids (PUFA) selectively suppress hepatic SREBP-1, but molecular mechanisms remain largely unknown. To gain insight into this regulation, we established in vivo reporter assays to assess the activities of Srebf1c transcription and proteolytic processing. Using these in vivo reporter assays, we showed that the primary mechanism for P...

  9. Predicting protein ligand binding motions with the conformation explorer

    Directory of Open Access Journals (Sweden)

    Flores Samuel C

    2011-10-01

    Full Text Available Abstract Background Knowledge of the structure of proteins bound to known or potential ligands is crucial for biological understanding and drug design. Often the 3D structure of the protein is available in some conformation, but binding the ligand of interest may involve a large scale conformational change which is difficult to predict with existing methods. Results We describe how to generate ligand binding conformations of proteins that move by hinge bending, the largest class of motions. First, we predict the location of the hinge between domains. Second, we apply an Euler rotation to one of the domains about the hinge point. Third, we compute a short-time dynamical trajectory using Molecular Dynamics to equilibrate the protein and ligand and correct unnatural atomic positions. Fourth, we score the generated structures using a novel fitness function which favors closed or holo structures. By iterating the second through fourth steps we systematically minimize the fitness function, thus predicting the conformational change required for small ligand binding for five well studied proteins. Conclusions We demonstrate that the method in most cases successfully predicts the holo conformation given only an apo structure.

  10. A homeodomain protein binds to. gamma. -globin gene regulatory sequences

    Energy Technology Data Exchange (ETDEWEB)

    Lavelle, D.; Ducksworth, J.; Eves, E.; Gomes, G.; Keller, M.; Heller, P.; DeSimone, J. (Univ. of Illinois, Chicago (United States) Veterans Administration Westside Medical Center, Chicago, IL (United States))

    1991-08-15

    Developmental regulation of {gamma}-globin gene expression probably occurs through developmental-stage-specific trans-acting factors able to promote the interaction of enhancer elements located in the far upstream locus control region with regulatory elements in the {gamma} gene promoters and 3{prime}{sup A}{gamma} enhancer located in close proximity to the genes. The authors have detected a nuclear protein in K562 and baboon fetal bone marrow nuclear extracts capable of binding to A+T-rich sequences in the locus control region, {gamma} gene promoter, and 3{prime} {sup A}{gamma} enhancer. SDS/polyacrylamide gel analysis of the purified K562 binding activity revealed a single protein of 87 kDa. A K562 cDNA clone was isolated encoding a {beta}-galactosidase fusion protein with a DNA binding specificity identical to that of the K562/fetal bone marrow nuclear protein. The cDNA clone encodes a homeodomain homologous to the Drosophila antennapedia protein.

  11. Treponema pallidum receptor binding proteins interact with fibronectin

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, K.M.; Baseman, J.B.; Alderete, J.F.

    1983-06-01

    Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp-2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or /sup 35/S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition.

  12. Treponema pallidum receptor binding proteins interact with fibronectin

    International Nuclear Information System (INIS)

    Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp-2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or 35S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition

  13. Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

    Science.gov (United States)

    Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

    2016-12-15

    In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins. PMID:27451201

  14. Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89.

    Science.gov (United States)

    Pleass, R J; Areschoug, T; Lindahl, G; Woof, J M

    2001-03-16

    Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function. PMID:11096107

  15. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the...

  16. Comparative study of methyl-CpG-binding domain proteins

    Directory of Open Access Journals (Sweden)

    Ropers H Hilger

    2003-01-01

    Full Text Available Abstract Background Methylation at CpG dinucleotides in genomic DNA is a fundamental epigenetic mechanism of gene expression control in vertebrates. Proteins with a methyl-CpG-binding domain (MBD can bind to single methylated CpGs and most of them are involved in transcription control. So far, five vertebrate MBD proteins have been described as MBD family members: MBD1, MBD2, MBD3, MBD4 and MECP2. Results We performed database searches for new proteins containing an MBD and identified six amino acid sequences which are different from the previously described ones. Here we present a comparison of their MBD sequences, additional protein motifs and the expression of the encoding genes. A calculated unrooted dendrogram indicates the existence of at least four different groups of MBDs within these proteins. Two of these polypeptides, KIAA1461 and KIAA1887, were only present as predicted amino acid sequences based on a partial human cDNA. We investigated their expression by Northern blot analysis and found transcripts of ~8 kb and ~5 kb respectively, in all eight normal tissues studied. Conclusions Eleven polypeptides with a MBD could be identified in mouse and man. The analysis of protein domains suggests a role in transcriptional regulation for most of them. The knowledge of additional existing MBD proteins and their expression pattern is important in the context of Rett syndrome.

  17. Polyamine binding to proteins in oat and Petunia protoplasts

    Science.gov (United States)

    Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

    1989-01-01

    Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

  18. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    Sumedha Roy; Shayoni Dutta; Kanika Khanna; Shruti Singla; Durai Sundar

    2012-07-01

    Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions −1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure–function relationships of the existing zinc finger–DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.

  19. Insulin-like growth factor binding proteins: a structural perspective

    Directory of Open Access Journals (Sweden)

    Briony eForbes

    2012-03-01

    Full Text Available Insulin-like growth factor binding proteins (IGFBP-1 to -6 bind insulin-like growth factors-I and -II (IGF-I and IGF-II with high affinity. These binding proteins maintain IGFs in the circulation and direct them to target tissues, where they promote cell growth, proliferation, differentiation and survival via the type 1 IGF receptor (IGF-1R. IGFBPs also interact with many other molecules, which not only influence their modulation of IGF action but also mediate IGF-independent activities that influence processes such as cell migration and apoptosis by influencing gene transcription.IGFBPs-1 to -6 are structurally similar proteins consisting of three distinct domains, N-terminal, Linker and C-terminal. There have been major advances in our understanding of IGFBP structure in the last decade and a half. While there is still no structure of an intact IGFBP to date, several structures of individual N- and C-domains have been solved. The structure of a complex of N-BP-4:IGF-I:C-BP-4 has also been solved, providing a detailed picture of the structural features of the IGF binding site and the mechanism of binding. Structural studies have also identified features important for interaction with extracellular matrix components and integrins. This review summarises structural studies reported so far and highlights features important for binding not only IGF but also other partners. It also highlights future directions in which structural studies will add to our knowledge of the role played by the IGFBP family in normal growth and development, as well as in disease.

  20. Stable Isotope Labeling Strategy for Protein-Ligand Binding Analysis in Multi-Component Protein Mixtures

    Science.gov (United States)

    DeArmond, Patrick D.; West, Graham M.; Huang, Hai-Tsang; Fitzgerald, Michael C.

    2011-03-01

    Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein-ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein-ligand binding interactions. The protocol utilizes an H{2/16}O2 and H{2/18}O2 labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein-ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the 18O/16O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique's ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein-protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).

  1. [Penicillin-binding proteins of various strains of Lactobacillus].

    Science.gov (United States)

    Griaznova, N S; Subbotina, N A; Beliavskaia, I V; Taisova, A S; Afonin, V I; Tiurin, M V; Shenderov, B A; Sazykina, Iu O; Navashin, S M

    1990-02-01

    Sensitivity of different species of Lactobacillus i.e. L. casei, L. plantarum, L. acidophillus, L. buchneri, L. jugurti and others to penicillins and cephalosporins of various generations was studied. Penicillin binding proteins (PBPs) of the Lactobacillus species were specified. It was shown that the number of PBPs depended on the Lactobacillus species. L. casei had the least number of PBPs (4) and L. brevis had the highest number of PBPs (11). Competition of 14C-benzylpenicillin with ampicillin, cefotaxime, ceftizoxime and cefoperazone for binding to separate PBPs in three strains of different Lactobacillus species was investigated. PMID:2110806

  2. Photoaffinity labelling of high affinity dopamine binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ross, G.M.; McCarry, B.E.; Mishra, R.K.

    1986-03-01

    A photoactive analogue of the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) has been synthesized and used to photoaffinity label dopamine binding proteins prepared from bovine caudate nucleus. N-(3-)N'-4-azidobenzamidol)-aminopropyl)-aminopropyl)-ADTN (AzB-AP-ADTN) was incubated with caudate membranes and irradiated with UV light. Membranes were then repeatedly washed by centrifugation to remove excess photolabel. A binding assay, using (/sup 3/H)-SCH 23390 (a D/sub 1/ specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked (/sup 3/H)-SCH 23390 binding in a dose-dependent manner. Scatchard analysis revealed a decrease in the B/sub max/, with no significant change in the K/sub d/, of (/sup 3/H)-SCH 23390 binding. Compounds which compete for D/sub 1/ receptor binding (such as dopamine, SKF 38393 or apomorphine), proteted the SCH 23390 binding site from inactivation. This data would suggest that the novel photoaffinity ligand, AzB-AP-ADTN, can covalently label the D/sub 1/ (adenylate cyclase linked) dopamine receptor.

  3. Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Mercurio, A M; Chung, S Y;

    1990-01-01

    Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from the...... isolated and used to elicit a rabbit antiserum. In immunostaining, both antisera reacted with the nuclei of cultured tumor cells. In tissue sections of human carcinoma, nuclear immunoreactivity was observed in the tumor cells in 40 of 42 cases examined. Adjacent normal epithelial tissue obtained from the...... presence of the homeobox transcript in human carcinoma was documented by in situ hybridization and RNase protection mapping. These results demonstrate that human cancer is associated with the expression of homeobox proteins. Such homeobox proteins, as well as other regulatory proteins, could be involved in...

  4. Poisson-Boltzmann Calculations of Nonspecific Salt Effects on Protein-Protein Binding Free Energies

    OpenAIRE

    Bertonati, Claudia; Honig, Barry; Alexov, Emil

    2007-01-01

    The salt dependence of the binding free energy of five protein-protein hetero-dimers and two homo-dimers/tetramers was calculated from numerical solutions to the Poisson-Boltzmann equation. Overall, the agreement with experimental values is very good. In all cases except one involving the highly charged lactoglobulin homo-dimer, increasing the salt concentration is found both experimentally and theoretically to decrease the binding affinity. To clarify the source of salt effects, the salt-dep...

  5. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins.

    OpenAIRE

    Zamore, P. D.; Williamson, J R; Lehmann, R.

    1997-01-01

    Translation of hunchback(mat) (hb[mat]) mRNA must be repressed in the posterior of the pre-blastoderm Drosophila embryo to permit formation of abdominal segments. This translational repression requires two copies of the Nanos Response Element (NRE), a 16-nt sequence in the hb[mat] 3' untranslated region. Translational repression also requires the action of two proteins: Pumilio (PUM), a sequence-specific RNA-binding protein; and Nanos, a protein that determines the location of repression. Bin...

  6. Detection of an endothelin-1-binding protein complex by low temperature SDS-PAGE

    International Nuclear Information System (INIS)

    We found that the complex of ET-1 and its binding protein was stable enough to be separated by SDS-PAGE when electrophoresis was run at a low temperature. Cross-linking was not necessary for the detection of 125I-ET-1 and its binding protein complex by autoradiography. This simple method could be used in qualitative (estimation of apparent molecular weight of ET-1 binding protein) and quantitative (determination of relative content of ET-binding protein) analysis of the ET-binding protein complex. ET-binding protein complexes of various animal species and organs were investigated by this method

  7. Phosphorylation of bovine interphotoreceptor retinoid-binding protein (IRBP)

    International Nuclear Information System (INIS)

    IRBP is the major soluble (glycolipo) protein of the interphotoreceptor matrix (IPM) and a putative intercellular retinoid-transport vehicle. The authors have now examined phosphorylation of proteins in a crude bovine IPM wash using γ-32P-ATP. SDS-polyacrylamide gel electrophoresis (PAGE) of IPM proteins showed several phosphorylated protein bands, one of them migrating in the same position as purified IRBP. When an aliquot of phosphorylated IPM proteins was incubated overnight with 3H-retinol and subjected to either size-exclusion or ion-exchange HPLC, a peak of 32P was observed in both cases which coincided with 3H-retinol binding and had a retention time identical to that of purified IRBP. When phosphorylated IPM was subjected to Con A Sepharose affinity chromatography and the 50mM methyl α-D-mannoside eluate chromatographed on ion-exchange HPLC, the 32P-peak was not present although a substantial amount of non-phosphorylated IRBP was recovered as assessed by SDS-PAGE and Western blotting. However, when the Con A Sepharose beads were dissolved in SDS and subjected to SDS-PAGE and Western blotting, a band of phosphorylated IRBP was observed, indicating that the phosphorylated IRBP was more tightly bound to the Con A Sepharose. The authors conclude that a fraction of IRBP can be phosphorylated by a yet to be characterized protein kinase and that the binding characteristics of IRBP are markedly altered by phosphorylation

  8. Crystal Structure of Human Retinoblastoma Binding Protein 9

    Energy Technology Data Exchange (ETDEWEB)

    Vorobiev, S.; Su, M; Seetharaman, J; Huang, Y; Chen, C; Maglaqui, M; Janjua, H; Montelione, G; Tong, L; et. al.

    2009-01-01

    As a step towards better integrating protein three-dimensional (3D) structural information in cancer systems biology, the Northeast Structural Genomics Consortium (NESG) (www.nesg.org) has constructed a Human Cancer Pathway Protein Interaction Network (HCPIN) by analysis of several classical cancer-associated signaling pathways and their physical protein-protein interactions. Many well-known cancer-associated proteins play central roles as hubs or bottlenecks in the HCPIN (http://nmr.cabm.rutgers.edu/hcpin). NESG has selected more than 1000 human proteins and protein domains from the HCPIN for sample production and 3D structure determination. The long-range goal of this effort is to provide a comprehensive 3D structure-function database for human cancer-associated proteins and protein complexes, in the context of their interaction networks. Human retinoblastoma binding protein 9 (RBBP9) is one of the HCPIN proteins targeted by NESG. RBBP9 was initially identified as the product of a new gene, Bog (for B5T over-expressed gene), in several transformed rat liver epithelial cell lines resistant to the growth-inhibitory effect of TGF-1 as well as in primary human liver tumors. RBBP9 contains the retinoblastoma (Rb) binding motif LxCxE in its sequence, and was shown to interact with Rb by yeast two-hybrid and coimmunoprecipitation experiments. Mutation of the Leu residue in this motif to Gln blocked the binding to Rb. RBBP9 can displace E2F1 from E2F1-Rb complexes, and over expression of RBBP9 overcomes TGF-1 induced growth arrest and results in transformation of rat liver epithelial cells leading to hepatoblastoma-like tumors in nude mice. RBBP9 may also play a role in cellular responses to chronic low dose radiation. A close homolog of RBBP9, sharing 93% amino acid sequence identity and also known as RBBP10, interacts with a protein with sua5-yciO-yrdC domains.

  9. Predicting the Impact of Missense Mutations on Protein-Protein Binding Affinity.

    Science.gov (United States)

    Li, Minghui; Petukh, Marharyta; Alexov, Emil; Panchenko, Anna R

    2014-04-01

    The crucial prerequisite for proper biological function is the protein's ability to establish highly selective interactions with macromolecular partners. A missense mutation that alters the protein binding affinity may cause significant perturbations or complete abolishment of the function, potentially leading to diseases. The availability of computational methods to evaluate the impact of mutations on protein-protein binding is critical for a wide range of biomedical applications. Here, we report an efficient computational approach for predicting the effect of single and multiple missense mutations on protein-protein binding affinity. It is based on a well-tested simulation protocol for structure minimization, modified MM-PBSA and statistical scoring energy functions with parameters optimized on experimental sets of several thousands of mutations. Our simulation protocol yields very good agreement between predicted and experimental values with Pearson correlation coefficients of 0.69 and 0.63 and root-mean-square errors of 1.20 and 1.90 kcal mol(-1) for single and multiple mutations, respectively. Compared with other available methods, our approach achieves high speed and prediction accuracy and can be applied to large datasets generated by modern genomics initiatives. In addition, we report a crucial role of water model and the polar solvation energy in estimating the changes in binding affinity. Our analysis also reveals that prediction accuracy and effect of mutations on binding strongly depends on the type of mutation and its location in a protein complex. PMID:24803870

  10. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    Science.gov (United States)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  11. The surface protein Shr of Streptococcus pyogenes binds heme and transfers it to the streptococcal heme-binding protein Shp

    OpenAIRE

    Lei Benfang; Liu Mengyao; Zhu Hui

    2008-01-01

    Abstract Background The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established. Results The objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, ...

  12. Human neutrophil calmodulin-binding proteins: identification of the calmodulin-dependent protein phosphatase

    International Nuclear Information System (INIS)

    The molecular events in linking neutrophil activation and ligand binding to specific membrane receptors are mediated in part by an increase in intracellular Ca2+. One mechanism by which Ca2+ may trigger neutrophil activation is through Ca2+/calmodulin (CaM)-regulated proteins and enzymes. To determine which Ca2+/CaM-regulated enzymes may be present in the neutrophil, they have used Western blotting techniques and 125I-CaM to identify neutrophil CaM-binding proteins. Eleven proteins with molecular weights ranging from 230K to 13.5K bound 125I-CaM in a Ca2+-dependent manner. One predominant region of 125I-Cam binding was to a 59K protein; a protein with an identical mobility was labeled by an antisera against brain CaM-dependent phosphatase. Ca2+-dependent phosphatase activity, which was inhibited by the CaM antagonist trifluoperazine, was detected in a neutrophil extract; a radioimmunoassay for the phosphatase indicated that it was present in the extract at approximately 0.2 μg/mg protein. Most of the CaM-binding proteins, including the 59K protein, were rapidly degraded upon lysis of the neutrophil. There was a close correlation between the degradation of the 59K protein and the loss of Ca2+-dependent phosphatase activity in the neutrophil extract. Thus, human neutrophils contain numerous CaM-binding proteins which are presumably Ca2+/calmodulin-regulated enzymes and proteins; the 59K protein is a CaM-dependent phosphatase

  13. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

  14. Glycosylation status of vitamin D binding protein in cancer patients

    OpenAIRE

    Rehder, Douglas S.; Nelson, Randall W.; Borges, Chad R.

    2009-01-01

    On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, co...

  15. Interactions of human mannose-binding protein with lipoteichoic acids.

    OpenAIRE

    Polotsky, V Y; Fischer, W; Ezekowitz, R A; Joiner, K A

    1996-01-01

    We explored the interaction of human recombinant mannose-binding protein and lipoteichoic acids (LTAs) by enzyme-linked immunosorbent assay. The best ligand was Micrococcus luteus lipomannan, followed by Enterococcus spp. LTA containing mono-, di-, and oligoglucosyl substituents. LTAs lacking terminal sugars (those of Streptococcus pyogenes and Staphylococcus aureus) or containing galactosyl substituents (those of Listeria spp. and Lactococcus spp.) were poor ligands. These results are consis...

  16. Vibrational Softening of a Protein on Ligand Binding

    Energy Technology Data Exchange (ETDEWEB)

    Balog, Erica [Semmelweis University, Budapest, Hungary; Perahia, David [Ecole Normale Superieure de Cachan, Cachan, France; Smith, Jeremy C [ORNL; Merzel, Franci [National Institute of Chemistry, Solvenia

    2011-01-01

    Neutron scattering experiments have demonstrated that binding of the cancer drug methotrexate softens the low-frequency vibrations of its target protein, dihydrofolate reductase (DHFR). Here, this softening is fully reproduced using atomic detail normal-mode analysis. Decomposition of the vibrational density of states demonstrates that the largest contributions arise from structural elements of DHFR critical to stability and function. Mode-projection analysis reveals an increase of the breathing-like character of the affected vibrational modes consistent with the experimentally observed increased adiabatic compressibility of the protein on complexation.

  17. Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

    Science.gov (United States)

    Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

    2016-05-12

    Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project. PMID:27043011

  18. Characterization of auxin-binding proteins from zucchini plasma membrane

    Science.gov (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  19. Architecture of the sugar binding sites in carbohydrate binding proteins--a computer modeling study.

    Science.gov (United States)

    Rao, V S; Lam, K; Qasba, P K

    1998-11-01

    Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity. PMID:9849627

  20. Development of computational methods for the prediction of protein structure, protein binding, and mutational effects using free energy calculations.

    OpenAIRE

    Becker, Caroline

    2014-01-01

    A molecular understanding of protein-protein or protein-ligand binding is of crucial importance for the design of proteins or ligands with defined binding characteristics. The comprehensive analysis of biomolecular binding and the coupled rational in silico design of protein-ligand interfaces requires both, accurate and computationally fast methods for the prediction of free energies. Accurate free energy methods usually involve atomistic molecular dynamics simulations that are computationall...

  1. SiteComp: a server for ligand binding site analysis in protein structures

    OpenAIRE

    Lin, Yingjie; Yoo, Seungyeul; Sanchez, Roberto

    2012-01-01

    Motivation: Computational characterization of ligand-binding sites in proteins provides preliminary information for functional annotation, protein design and ligand optimization. SiteComp implements binding site analysis for comparison of binding sites, evaluation of residue contribution to binding sites and identification of sub-sites with distinct molecular interaction properties.

  2. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    Science.gov (United States)

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  3. Cloning and characterization of a novel hepatitis B virus core binding protein C12

    Institute of Scientific and Technical Information of China (English)

    Yin-Ying Lu; Jun Cheng; Yong-Ping Yang; Yan Liu; Lin Wang; Ke Li; Ling-Xia Zhang

    2005-01-01

    AIM: To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS: HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade)containing X-α-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating.pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein.RESULTS: C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray,of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function

  4. Functional interactions between polypyrimidine tract binding protein and PRI peptide ligand containing proteins.

    Science.gov (United States)

    Coelho, Miguel B; Ascher, David B; Gooding, Clare; Lang, Emma; Maude, Hannah; Turner, David; Llorian, Miriam; Pires, Douglas E V; Attig, Jan; Smith, Christopher W J

    2016-08-15

    Polypyrimidine tract binding protein (PTBP1) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that plays roles in most stages of the life-cycle of pre-mRNA and mRNAs in the nucleus and cytoplasm. PTBP1 has four RNA binding domains of the RNA recognition motif (RRM) family, each of which can bind to pyrimidine motifs. In addition, RRM2 can interact via its dorsal surface with proteins containing short peptide ligands known as PTB RRM2 interacting (PRI) motifs, originally found in the protein Raver1. Here we review our recent progress in understanding the interactions of PTB with RNA and with various proteins containing PRI ligands. PMID:27528752

  5. The human enhancer-binding protein Gata3 binds to several T-cell receptor regulatory elements.

    OpenAIRE

    Marine, J; Winoto, A

    1991-01-01

    The tissue-specific developmental regulation of the alpha, beta, gamma and delta T-cell antigen receptor (TCR) genes is controlled by the corresponding distinct enhancers and their enhancer-binding proteins. To find a common TCR regulatory element, we have studied the ability of the newly described enhancer-binding protein Gata3 to bind to the sequence motif (A/T)GATA(G/A) shared between enhancer elements of all four TCR genes. Gata3 was shown in the chicken to be an enhancer-binding protein ...

  6. Cellular inhibitor of apoptosis protein-1 (cIAP1) can regulate E2F1 transcription factor-mediated control of cyclin transcription.

    Science.gov (United States)

    Cartier, Jessy; Berthelet, Jean; Marivin, Arthur; Gemble, Simon; Edmond, Valérie; Plenchette, Stéphanie; Lagrange, Brice; Hammann, Arlette; Dupoux, Alban; Delva, Laurent; Eymin, Béatrice; Solary, Eric; Dubrez, Laurence

    2011-07-29

    The inhibitor of apoptosis protein cIAP1 (cellular inhibitor of apoptosis protein-1) is a potent regulator of the tumor necrosis factor (TNF) receptor family and NF-κB signaling pathways in the cytoplasm. However, in some primary cells and tumor cell lines, cIAP1 is expressed in the nucleus, and its nuclear function remains poorly understood. Here, we show that the N-terminal part of cIAP1 directly interacts with the DNA binding domain of the E2F1 transcription factor. cIAP1 dramatically increases the transcriptional activity of E2F1 on synthetic and CCNE promoters. This function is not conserved for cIAP2 and XIAP, which are cytoplasmic proteins. Chromatin immunoprecipitation experiments demonstrate that cIAP1 is recruited on E2F binding sites of the CCNE and CCNA promoters in a cell cycle- and differentiation-dependent manner. cIAP1 silencing inhibits E2F1 DNA binding and E2F1-mediated transcriptional activation of the CCNE gene. In cells that express a nuclear cIAP1 such as HeLa, THP1 cells and primary human mammary epithelial cells, down-regulation of cIAP1 inhibits cyclin E and A expression and cell proliferation. We conclude that one of the functions of cIAP1 when localized in the nucleus is to regulate E2F1 transcriptional activity. PMID:21653699

  7. Bile salt recognition by human liver fatty acid binding protein.

    Science.gov (United States)

    Favretto, Filippo; Santambrogio, Carlo; D'Onofrio, Mariapina; Molinari, Henriette; Grandori, Rita; Assfalg, Michael

    2015-04-01

    Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 μm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder. PMID:25639618

  8. Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins.

    Science.gov (United States)

    Maeda, Kenji; Anand, Kanchan; Chiapparino, Antonella; Kumar, Arun; Poletto, Mattia; Kaksonen, Marko; Gavin, Anne-Claude

    2013-09-12

    The internal organization of eukaryotic cells into functionally specialized, membrane-delimited organelles of unique composition implies a need for active, regulated lipid transport. Phosphatidylserine (PS), for example, is synthesized in the endoplasmic reticulum and then preferentially associates--through mechanisms not fully elucidated--with the inner leaflet of the plasma membrane. Lipids can travel via transport vesicles. Alternatively, several protein families known as lipid-transfer proteins (LTPs) can extract a variety of specific lipids from biological membranes and transport them, within a hydrophobic pocket, through aqueous phases. Here we report the development of an integrated approach that combines protein fractionation and lipidomics to characterize the LTP-lipid complexes formed in vivo. We applied the procedure to 13 LTPs in the yeast Saccharomyces cerevisiae: the six Sec14 homology (Sfh) proteins and the seven oxysterol-binding homology (Osh) proteins. We found that Osh6 and Osh7 have an unexpected specificity for PS. In vivo, they participate in PS homeostasis and the transport of this lipid to the plasma membrane. The structure of Osh6 bound to PS reveals unique features that are conserved among other metazoan oxysterol-binding proteins (OSBPs) and are required for PS recognition. Our findings represent the first direct evidence, to our knowledge, for the non-vesicular transfer of PS from its site of biosynthesis (the endoplasmic reticulum) to its site of biological activity (the plasma membrane). We describe a new subfamily of OSBPs, including human ORP5 and ORP10, that transfer PS and propose new mechanisms of action for a protein family that is involved in several human pathologies such as cancer, dyslipidaemia and metabolic syndrome. PMID:23934110

  9. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue.

    Science.gov (United States)

    Chen, Wentao; Dong, Jiajia; Plate, Lars; Mortenson, David E; Brighty, Gabriel J; Li, Suhua; Liu, Yu; Galmozzi, Andrea; Lee, Peter S; Hulce, Jonathan J; Cravatt, Benjamin F; Saez, Enrique; Powers, Evan T; Wilson, Ian A; Sharpless, K Barry; Kelly, Jeffery W

    2016-06-15

    Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-molecule ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis experiments, provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function. PMID:27191344

  10. Buffer Interference with Protein Dynamics: A Case Study on Human Liver Fatty Acid Binding Protein

    OpenAIRE

    Long, Dong; Yang, Daiwen

    2009-01-01

    Selection of suitable buffer types is often a crucial step for generating appropriate protein samples for NMR and x-ray crystallographic studies. Although the possible interaction between MES buffer (2-(N-morpholino)ethanesulfonic acid) and proteins has been discussed previously, the interaction is usually thought to have no significant effects on the structures of proteins. In this study, we demonstrate the direct, albeit weak, interaction between MES and human liver fatty acid binding prote...

  11. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    Science.gov (United States)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-03-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  12. Local Unfolding of Fatty Acid Binding Protein to Allow Ligand Entry for Binding.

    Science.gov (United States)

    Xiao, Tianshu; Fan, Jing-Song; Zhou, Hu; Lin, Qingsong; Yang, Daiwen

    2016-06-01

    Fatty acid binding proteins are responsible for the transportation of fatty acids in biology. Despite intensive studies, the molecular mechanism of fatty acid entry to and exit from the protein cavity is still unclear. Here a cap-closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the helical cap is locked to the β-barrel by a disulfide linkage. Structure determination shows that this variant adopts a closed conformation, but still uptakes fatty acids. Stopped-flow experiments indicate that a rate-limiting step exists before the ligand association and this step corresponds to the conversion of the closed form to the open one. NMR relaxation dispersion and H-D exchange data demonstrate the presence of two excited states: one is native-like, but the other adopts a locally unfolded structure. Local unfolding of helix 2 generates an opening for ligands to enter the protein cavity, and thus controls the ligand association rate. PMID:27105780

  13. Interplay between binding affinity and kinetics in protein-protein interactions.

    Science.gov (United States)

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. PMID:27018856

  14. Computational analysis of HIV-1 protease protein binding pockets.

    Science.gov (United States)

    Ko, Gene M; Reddy, A Srinivas; Kumar, Sunil; Bailey, Barbara A; Garg, Rajni

    2010-10-25

    Mutations that arise in HIV-1 protease after exposure to various HIV-1 protease inhibitors have proved to be a difficult aspect in the treatment of HIV. Mutations in the binding pocket of the protease can prevent the protease inhibitor from binding to the protein effectively. In the present study, the crystal structures of 68 HIV-1 proteases complexed with one of the nine FDA approved protease inhibitors from the Protein Data Bank (PDB) were analyzed by (a) identifying the mutational changes with the aid of a developed mutation map and (b) correlating the structure of the binding pockets with the complexed inhibitors. The mutations of each crystal structure were identified by comparing the amino acid sequence of each structure against the HIV-1 wild-type strain HXB2. These mutations were visually presented in the form of a mutation map to analyze mutation patterns corresponding to each protease inhibitor. The crystal structure mutation patterns of each inhibitor (in vitro) were compared against the mutation patterns observed in in vivo data. The in vitro mutation patterns were found to be representative of most of the major in vivo mutations. We then performed a data mining analysis of the binding pockets from each crystal structure in terms of their chemical descriptors to identify important structural features of the HIV-1 protease protein with respect to the binding conformation of the HIV-1 protease inhibitors. Data mining analysis is performed using several classification techniques: Random Forest (RF), linear discriminant analysis (LDA), and logistic regression (LR). We developed two hybrid models, RF-LDA and RF-LR. Random Forest is used as a feature selection proxy, reducing the descriptor space to a few of the most relevant descriptors determined by the classifier. These descriptors are then used to develop the subsequent LDA, LR, and hierarchical classification models. Clustering analysis of the binding pockets using the selected descriptors used to

  15. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  16. Liver fatty acid-binding protein binds monoacylglycerol in vitro and in mouse liver cytosol.

    Science.gov (United States)

    Lagakos, William S; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E; Storch, Judith

    2013-07-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803-G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP(-/-) mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  17. Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol*

    Science.gov (United States)

    Lagakos, William S.; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E.; Storch, Judith

    2013-01-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  18. Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein

    DEFF Research Database (Denmark)

    Salanti, Ali; Clausen, Thomas M.; Agerbæk, Mette Ø.;

    2015-01-01

    Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can...... be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds...... strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification....

  19. The clinical significance of fatty acid binding proteins

    Directory of Open Access Journals (Sweden)

    Barbara Choromańska

    2011-11-01

    Full Text Available Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs that bind long-chain fatty acids (LCFA, and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum. So far, nine types of these proteins have been described, and their name refers to the place in which they were first identified or where they can be found in the greatest concentration. The most important FABPs were isolated from the liver (L-FABP, heart (H-FABP, intestine (I-FABP, brain (B-FABP, epidermis (E-FABP and adipocytes (A-FABP. Determination of H-FABP is used in the diagnosis of myocardial infarction, and L-FABP in kidney lesions of different etiologies. It is postulated that FABPs play an important role in the pathogenesis of metabolic diseases. Elevated levels of A-FABP have been found in the pericardial fat tissue and were associated with cardiac dysfunction in obese people. A rise in A-FABP has been observed in patients with type II diabetes. I-FABP is known as a marker of cell damage in the small intestine. Increased concentration of B-FABP has been associated with human brain tumors such as glioblastoma and astrocytoma, as well as with neurodegenerative diseases (Alzheimer’s, Parkinson’s and other disorders of cognitive function. The aim of this work was to present current data on the clinical significance of fatty acid binding proteins.

  20. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

  1. Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein

    Science.gov (United States)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis. Copyright 2000 Academic Press.

  2. Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

    Directory of Open Access Journals (Sweden)

    Bradley Michael E

    2006-02-01

    Full Text Available Abstract Background When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1 multiple sequence alignments, 2 mapping of alignment sites to crystal structure sites, 3 phylogenetic trees, 4 inferred ancestral sequences at internal tree nodes, and 5 amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural

  3. In vitro binding of selenium by rat liver mitochondrial selenium-binding protein

    International Nuclear Information System (INIS)

    Last year the authors reported that upon freezing and thawing mitochondria from rats injected with [75Se]Na2SeO3 (75Se-selenite), a 75Se-binding protein (SeBP) was released. They have studied further in vitro labelling of SeBP. This matrix protein was labelled in vitro when lysed mitochondria (containing non-matrix material) were incubated with 75Se-selenite but not when matrix material alone was incubated with 75Se-selenite. Thus, there are one or more promoters of in vitro SeBP labelling in the non-matrix fraction. SeBP was also labelled in vitro when 75Se-selenite was added to matrix alone and dialyzed. Dialysis tubing, and not the dialysis process, promoted labelling by affecting SeBP and not by affecting 75Se-selenite. Labelling did not occur when matrix alone and 75Se-selenite were incubated (not dialyzed) in a glass test tube but did occur in a polystyrene test tube. They hypothesize that non-covalent interactions occur between SeBP and dialysis tubing or polystyrene that expose Se binding sites on the protein. A similar mechanism involving mitochondrial non-matrix material may function in vivo. Non-denaturing disc gel electrophoresis of partially purified SeBP labelled in vivo or in vitro suggested that the same protein was labelled in both conditions. Using in vitro binding techniques, SeBP was also found in sheep liver mitochondrial matrix. This supports the theory that SeBP is important in Se metabolism

  4. Functional zinc-binding motifs in enzymes and DNA-binding proteins.

    Science.gov (United States)

    Vallee, B L; Auld, D S

    1992-01-01

    Zinc is now known to be an integral component of a large number and variety of enzymes and proteins involved in virtually all aspects of metabolism, thus accounting for the fact that this element is essential for growth and development. The chemistry of zinc, superficially bland, in reality has turned out to be ideally appropriate and versatile for the unexpected development of multiple and unique chemical structures which biology has used for specific life processes. The present discussion will centre on those distinctive zinc-binding motifs that are critical both to enzyme function and the expression of the genetic message. X-Ray diffraction structure determination of 15 zinc enzymes belonging to IUB classes I-IV provide absolute standards of reference for the identity and nature of zinc ligands in their families. Three types of zinc enzyme binding motifs emerge through analysis of these: catalytic, coactive or cocatalytic, and structural. In contrast to zinc enzymes virtually all DNA-binding proteins contain multiple zinc atoms. With the availability of NMR and X-ray structure analyses three distinct motifs now emerge for those: zinc fingers, twists and clusters. PMID:1290939

  5. The Movable Type Method Applied to Protein-Ligand Binding

    Science.gov (United States)

    Zheng, Zheng; Ucisik, Melek N.; Merz, Kenneth M.

    2013-01-01

    Accurately computing the free energy for biological processes like protein folding or protein-ligand association remains a challenging problem. Both describing the complex intermolecular forces involved and sampling the requisite configuration space make understanding these processes innately difficult. Herein, we address the sampling problem using a novel methodology we term “movable type”. Conceptually it can be understood by analogy with the evolution of printing and, hence, the name movable type. For example, a common approach to the study of protein-ligand complexation involves taking a database of intact drug-like molecules and exhaustively docking them into a binding pocket. This is reminiscent of early woodblock printing where each page had to be laboriously created prior to printing a book. However, printing evolved to an approach where a database of symbols (letters, numerals, etc.) was created and then assembled using a movable type system, which allowed for the creation of all possible combinations of symbols on a given page, thereby, revolutionizing the dissemination of knowledge. Our movable type (MT) method involves the identification of all atom pairs seen in protein-ligand complexes and then creating two databases: one with their associated pairwise distant dependent energies and another associated with the probability of how these pairs can combine in terms of bonds, angles, dihedrals and non-bonded interactions. Combining these two databases coupled with the principles of statistical mechanics allows us to accurately estimate binding free energies as well as the pose of a ligand in a receptor. This method, by its mathematical construction, samples all of configuration space of a selected region (the protein active site here) in one shot without resorting to brute force sampling schemes involving Monte Carlo, genetic algorithms or molecular dynamics simulations making the methodology extremely efficient. Importantly, this method explores the

  6. Zinc ions bind to and inhibit activated protein C

    DEFF Research Database (Denmark)

    Zhu, Tianqing; Ubhayasekera, Wimal; Nickolaus, Noëlle;

    2010-01-01

    Zn2+ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K I was 13 +/- 2 microM. k cat was...... seven fold decreased whereas K M was unaffected in the presence of 10 microM Zn2+. The inhibitory effect of Zn2+ on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn2+ with APC was accompanied by a reversible...... fold enhanced, presumably due to the Ca2+-induced conformational change affecting the conformation of the Zn2+-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca2+. Comparisons of sequences and structures suggested several possible sites for zinc binding...

  7. A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

    Directory of Open Access Journals (Sweden)

    Brian Krumm

    Full Text Available Interleukin 18 (IL18 is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding

  8. Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins.

    Science.gov (United States)

    Abu-Elneel, K; Robinson, D R; Drew, M E; Englund, P T; Shlomai, J

    2001-05-14

    Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication. PMID:11352934

  9. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

    Directory of Open Access Journals (Sweden)

    Kamala Boonyodying

    2012-06-01

    Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

  10. The MTA family proteins as novel histone H3 binding proteins

    Directory of Open Access Journals (Sweden)

    Wu Meng

    2013-01-01

    Full Text Available Abstract Background The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s responsible for specific binding of H3 by NURD has not been defined. Results In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Conclusions Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail.

  11. Using persistent homology and dynamical distances to analyze protein binding.

    Science.gov (United States)

    Kovacev-Nikolic, Violeta; Bubenik, Peter; Nikolić, Dragan; Heo, Giseon

    2016-03-01

    Persistent homology captures the evolution of topological features of a model as a parameter changes. The most commonly used summary statistics of persistent homology are the barcode and the persistence diagram. Another summary statistic, the persistence landscape, was recently introduced by Bubenik. It is a functional summary, so it is easy to calculate sample means and variances, and it is straightforward to construct various test statistics. Implementing a permutation test we detect conformational changes between closed and open forms of the maltose-binding protein, a large biomolecule consisting of 370 amino acid residues. Furthermore, persistence landscapes can be applied to machine learning methods. A hyperplane from a support vector machine shows the clear separation between the closed and open proteins conformations. Moreover, because our approach captures dynamical properties of the protein our results may help in identifying residues susceptible to ligand binding; we show that the majority of active site residues and allosteric pathway residues are located in the vicinity of the most persistent loop in the corresponding filtered Vietoris-Rips complex. This finding was not observed in the classical anisotropic network model. PMID:26812805

  12. Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

    DEFF Research Database (Denmark)

    Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M; Wallace, Mairi; Samejima, Itaru; Taylor, Martin S; Gordon, Colin

    2003-01-01

    Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also foun...

  13. DnaT is a PriC-binding protein.

    Science.gov (United States)

    Huang, Chien-Chih; Huang, Cheng-Yang

    2016-09-01

    DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT-PriC changed from red to purple, which indicated the protein-protein interactions due to gold nanoparticle aggregate. In addition, this DnaT-PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10(-8) M. These results constitute a pioneering study of the DnaT-PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC-SSB-DNA tricomplex and regulates the PriC-dependent replication restart. PMID:27387236

  14. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    Directory of Open Access Journals (Sweden)

    Ilse Jongerius

    Full Text Available Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH, a negative regulator of the complement system, to its surface via fH binding protein (fHbp, providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.

  15. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    Science.gov (United States)

    Jongerius, Ilse; Lavender, Hayley; Tan, Lionel; Ruivo, Nicola; Exley, Rachel M; Caesar, Joseph J E; Lea, Susan M; Johnson, Steven; Tang, Christoph M

    2013-01-01

    Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups. PMID:23935503

  16. The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins.

    OpenAIRE

    C. Du; Nickerson, K W

    1996-01-01

    Brush border membrane vesicles from larvae of the tobacco hornworm, Manduca sexta, contain protein bands of 85 and 120 kDa which react directly with streptavidin conjugated to alkaline phosphatase. The binding could be prevented either by including 10 microM biotin in the reaction mixture or by prior incubation of the brush border membrane vesicles with an activated 60- to 65-kDa toxin from Bacillus thuringiensis HD-73. The ability of B. thuringiensis toxins to recognize biotin-containing pro...

  17. Structural and binding properties of two paralogous fatty acid binding proteins of Taenia solium metacestode.

    Directory of Open Access Journals (Sweden)

    Seon-Hee Kim

    Full Text Available BACKGROUND: Fatty acid (FA binding proteins (FABPs of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs of Taenia solium metacestode (TsM, a causative agent of neurocysticercosis (NC, shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2, which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15-95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1 and 8.4 (TsMFABP2. Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]aminoundecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions

  18. Unusual Heme Binding in the Bacterial Iron Response Regulator Protein (Irr): Spectral Characterization of Heme Binding to Heme Regulatory Motif

    OpenAIRE

    Ishikawa, Haruto; Nakagaki, Megumi; Bamba, Ai; Uchida, Takeshi; Hori, Hiroshi; O'Brian, Mark R.; Iwai, Kazuhiro; Ishimori, Koichiro

    2011-01-01

    We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single “heme-regulatory motif”, HRM, and plays a key role in the iron homeostasis of a nitrogen fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where 29Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside...

  19. Roles of RNA-Binding Proteins in DNA Damage Response.

    Science.gov (United States)

    Kai, Mihoko

    2016-01-01

    Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, "sensor" proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)'s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as "liquid-demixing"). Among the

  20. Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

    Directory of Open Access Journals (Sweden)

    Andreas Bitter

    2015-01-01

    Full Text Available Background/Aims: Sterol regulatory element-binding protein (SREBP 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

  1. Protein-protein binding before and after photo-modification of albumin

    Science.gov (United States)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  2. Identification of Actin-Binding Proteins from Maize Pollen

    Energy Technology Data Exchange (ETDEWEB)

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  3. A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

    KAUST Repository

    Chen, Peng

    2015-12-03

    Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

  4. Periplasmic Binding Proteins in Thermophiles: Characterization and Potential Application of an Arginine-Binding Protein from Thermotoga maritima: A Brief Thermo-Story

    Directory of Open Access Journals (Sweden)

    Sabato D'Auria

    2013-02-01

    Full Text Available Arginine-binding protein from the extremophile Thermotoga maritima is a 27.7 kDa protein possessing the typical two-domain structure of the periplasmic binding proteins family. The protein is characterized by a very high specificity and affinity to bind to arginine, also at high temperatures. Due to its features, this protein could be taken into account as a potential candidate for the design of a biosensor for arginine. It is important to investigate the stability of proteins when they are used for biotechnological applications. In this article, we review the structural and functional features of an arginine-binding protein from the extremophile Thermotoga maritima with a particular eye on its potential biotechnological applications.

  5. A1C test

    Science.gov (United States)

    HbA1C test; Glycated hemoglobin test; Glycosylated hemoglobin test; Hemoglobin glycosylated test; Glycohemoglobin test ... have recently eaten does not affect the A1C test, so you do not need to fast to ...

  6. Protein interactions and ligand binding: From protein subfamilies to functional specificity

    OpenAIRE

    Rausell, A.; de Juan, D.; Pazos, F; Valencia, A.

    2010-01-01

    The divergence accumulated during the evolution of protein families translates into their internal organization as subfamilies, and it is directly reflected in the characteristic patterns of differentially conserved residues. These specifically conserved positions in protein subfamilies are known as “specificity determining positions” (SDPs). Previous studies have limited their analysis to the study of the relationship between these positions and ligand-binding specificity, demonstrating sign...

  7. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    Science.gov (United States)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  8. Cobalamin binding proteins in patients with HIV infection

    International Nuclear Information System (INIS)

    P-Cobalamins have been reported to be decreased in patients with HIV infection. Because of this, we found it of interest to examine both cobalamin-saturated binding proteins (holo-transcobalamin, holo-TC and holo-haptocorrin, holo-HC) and cobalamin unsaturated binding proteins (apo-transcobalamin, apo-TC and apo-haptocorrin, apo-HC). The results are given as range and (median). Eighteen male HIV-infected patients with plasma cobalamins below 200 pmol/l were studied. We found low concentrations of holo-TC (37-88(47.5)pmol/l) and holo-HC (64-184(135.3)pmol/l). The concentration of apo-TC and apo-HC was increased (480-1730(1025)pmol/l; 70-800(235)pmol/l). It is concluded that, in HIV-infected patients, low plasma cobalamin does not reflect a low concentration of transcobalamin or haptocorrin. In 20 HIV-infected patients and 31 patients with malignant haematological diseases, the TC isopeptide patterns were determined. In the HIV group, an increased frequency of TC isopeptide X was found and the overall distribution of TC isopeptides was significantly different from the reference population (p<0.05). There was no difference between the group of patients with malignant haematological diseases and the reference group. (au)

  9. Localization of Cellular Retinol-Binding Protein and Retinol-Binding Protein in Cells Comprising the Blood-Brain Barrier of Rat and Human

    Science.gov (United States)

    MacDonald, Paul N.; Bok, Dean; Ong, David E.

    1990-06-01

    Brain is not generally recognized as an organ that requiries vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the endothelial cells of the brain microvasculature and within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

  10. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    Energy Technology Data Exchange (ETDEWEB)

    MacDonald, P.N.; Ong, D.E. (Vanderbilt Univ., Nashville, TN (USA)); Bok, D. (Univ. of California, Los Angeles (USA))

    1990-06-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

  11. Protein kinase A binds and activates heat shock factor 1.

    Directory of Open Access Journals (Sweden)

    Ayesha Murshid

    Full Text Available BACKGROUND: Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1, a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. METHODOLOGY/PRINCIPAL FINDINGS: In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA. HSF1 binds avidly to the catalytic subunit of PKA, (PKAcα and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320, both in vitro and in vivo. Intracellular PKAcα levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1 and activate hsp70.1 after stress. Reduction in PKAcα levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, null mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. CONCLUSIONS: These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease.

  12. Plant Cytosolic Acyl-CoA-Binding Proteins.

    Science.gov (United States)

    Ye, Zi-Wei; Chye, Mee-Len

    2016-01-01

    A gene family encoding six members of acyl-CoA-binding proteins (ACBP) exists in Arabidopsis and they are designated as AtACBP1-AtACBP6. They have been observed to play pivotal roles in plant lipid metabolism, consistent to the abilities of recombinant AtACBP in binding different medium- and long-chain acyl-CoA esters in vitro. While AtACBP1 and AtACBP2 are membrane-associated proteins with ankyrin repeats and AtACBP3 contains a signaling peptide for targeting to the apoplast, AtACBP4, AtACBP5 and AtACBP6 represent the cytosolic forms in the AtACBP family. They were verified to be subcellularly localized in the cytosol using diverse experimental methods, including cell fractionation followed by western blot analysis, immunoelectron microscopy and confocal laser-scanning microscopy using autofluorescence-tagged fusions. AtACBP4 (73.2 kDa) and AtACBP5 (70.1 kDa) are the largest, while AtACBP6 (10.4 kDa) is the smallest. Their binding affinities to oleoyl-CoA esters suggested that they can potentially transfer oleoyl-CoA esters from the plastids to the endoplasmic reticulum, facilitating the subsequent biosynthesis of non-plastidial membrane lipids in Arabidopsis. Recent studies on ACBP, extended from a dicot (Arabidopsis) to a monocot, revealed that six ACBP are also encoded in rice (Oryza sativa). Interestingly, three small rice ACBP (OsACBP1, OsACBP2 and OsACBP3) are present in the cytosol in comparison to one (AtACBP6) in Arabidopsis. In this review, the combinatory and distinct roles of the cytosolic AtACBP are discussed, including their functions in pollen and seed development, light-dependent regulation and substrate affinities to acyl-CoA esters. PMID:26662549

  13. Electrophilicities and Protein Covalent Binding of Demethylation Metabolites of Colchicine.

    Science.gov (United States)

    Guo, Xiucai; Lin, Dongju; Li, Weiwei; Wang, Kai; Peng, Ying; Zheng, Jiang

    2016-03-21

    Colchicine, an alkaloid existing in plants of Liliaceous colchicum, has been widely used in the treatment of gout and familial Mediterranean fever. The administration of colchicine was found to cause liver injury in humans. The mechanisms of colchicine-induced liver toxicity remain unknown. The objectives of this study were to determine the electrophilicities of demethylation metabolites of colchicine and investigate the protein adductions derived from the reactive metabolites of colchicine. Four demethylated colchicine (1-, 2-, 3-, and 10-DMCs), namely, M1-M4, were detected in colchicine-fortified microsomal incubations. Four N-acetyl cysteine (NAC) conjugates (M5-M8) derived from colchicine were detected in the microsomes in the presence of NAC. M5 and M6 were derived from 10-DMC. M7 resulted from the reaction of 2-DMC or 3-DMC with NAC, and M8 originated from 10-DMC. Microsomal protein covalent binding was observed after exposure to colchicine. Two cysteine adducts (CA-1 and CA-2) derived from 10-DMC were found in proteolytically digested microsomal protein samples after incubation with colchicine. The findings allow us to define the chemical property of demethylation metabolites of colchicine and the interaction between protein and the reactive metabolites of colchicine generated in situ. PMID:26845511

  14. Tannin-binding salivary proteins in three captive rhinoceros species.

    Science.gov (United States)

    Clauss, Marcus; Gehrke, Janin; Hatt, Jean-Michel; Dierenfeld, Ellen S; Flach, Edmund J; Hermes, Robert; Castell, Johanna; Streich, W Juergen; Fickel, Joerns

    2005-01-01

    Tannin-binding salivary proteins (TBSP) are considered to be counter-defences acquired in the course of evolution by animals whose natural forage contains such tannins. As tannins mostly occur in browse material but not in grasses, it is assumed that grazers do not have a need for TBSP. Whereas it has been shown in several non-ungulate species that TBSP can be induced by dietary tannins, their presence or absence in ungulates has, so far, been shown to be a species-specific characteristic independent of dietary manipulations. We investigated saliva from three rhinoceros species from zoological gardens fed comparable, conventional zoo diets. As expected, saliva from white rhinoceroses (Ceratotherum simum, grazer) had lower tannin-binding capacities than that from black rhinoceroses (Diceros bicornis, browser). Surprisingly, however, Indian rhinoceroses (Rhinoceros unicornis), commonly regarded as grazers as well, displayed the highest tannin-binding capacities of the three species investigated. It is speculated that this discrepancy might be a result of an evolutionarily recent switch to a grass-dominated diet in Indian rhinoceroses, and that the black rhinoceros, which is closer related to the white rhinoceros than the Indian species, has evolved an inducible mechanism of TBSP production. In separate trials during which the tannin content of the diets of black rhinoceroses was increased by the addition of either tannic acid or quebracho, the tannin-binding capacity of black rhinoceros saliva was increased to levels within the same range as that of Indian rhinoceroses on the conventional diets. While induction trials in white and Indian rhinoceroses remain to be performed for a full understanding of salivary anti-tannin defence in rhinoceroses, these results are the first report of an induced salivary response to increased dietary tannin levels in an ungulate species. PMID:15664314

  15. XAS and Pulsed EPR Studies of the Copper Binding Site in Riboflavin Binding Protein

    Energy Technology Data Exchange (ETDEWEB)

    Smith,S.; Bencze, K.; Wasiukanis, K.; Benore-Parsons, T.; Stemmler, T.

    2008-01-01

    Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 Angstroms, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a CuO3N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.

  16. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  17. Enterocyte Fatty Acid Binding Proteins (FABPs): Different Functions of Liver- and Intestinal- FABPs in the Intestine

    OpenAIRE

    Gajda, Angela M.; Storch, Judith

    2014-01-01

    Fatty acid binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both Liver- (LFABP; FABP1) and Intestinal-fatty acid binding proteins (IFABP; FABP2) are expressed. These proteins display high affinity binding for long chain fatty acids (FA) and other hydrophobic ligands, thus they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences ...

  18. Effect of Protein Binding on the Pharmacological Activity of Highly Bound Antibiotics▿

    OpenAIRE

    Schmidt, Stephan; Röck, Katharina; Sahre, Martina; Burkhardt, Olaf; Brunner, Martin; Lobmeyer, Maximilian T.; Derendorf, Hartmut

    2008-01-01

    During antibiotic drug development, media are frequently spiked with either serum/plasma or protein supplements to evaluate the effect of protein binding. Usually, previously reported serum or plasma protein binding values are applied in the analysis. The aim of this study was to evaluate this approach by experimentally measuring free, unbound concentrations for antibiotics with reportedly high protein binding and their corresponding antimicrobial activities in media containing commonly used ...

  19. Acquisition of heme iron by Neisseria meningitidis does not involve meningococcal transferrin-binding proteins.

    Science.gov (United States)

    Martel, N; Lee, B C

    1994-02-01

    Similarities in size between hemin-binding protein 1 (HmBP1) and transferrin-binding protein 1 (TBP1) of Neisseria meningitidis suggest that these proteins are functionally homologous. However, a meningococcal mutant lacking the transferrin-binding proteins retained the capacity to acquire iron from heme and hemoglobin. In immunoblots, hyperimmune polyclonal antiserum against TBP1 did not react with HmBP1. PMID:8300227

  20. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

    Energy Technology Data Exchange (ETDEWEB)

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M.F.; Downes, C. Peter; Batty, Ian H. (Toronto); (Dundee)

    2012-10-16

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

  1. A single rainbow trout cobalamin-binding protein stands in for three human binders

    DEFF Research Database (Denmark)

    Greibe, Eva; Fedosov, Sergey; Sorensen, Boe S; Højrup, Peter; Poulsen, Steen Seier; Nexo, Ebba

    2012-01-01

    Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding pr...

  2. Binding of the human papillomavirus E1 origin-recognition protein is regulated through complex formation with the E2 enhancer-binding protein.

    OpenAIRE

    Frattini, M G; Laimins, L A

    1994-01-01

    The papillomavirus E1 and E2 proteins form heteromeric complexes and individually bind specific sequences within the viral origin of replication. The mechanism by which these proteins are recruited to the origin and the role of the E1/E2 complex in replication remain undefined. To examine the interplay of these replication proteins, we have analyzed the binding of human papillomavirus (HPV) type 31b E1 and E2 proteins to the origin of replication. Binding of E1 to the origin was increased by ...

  3. APPLICATION OF IMMUNOGLOBULIN-BINDING PROTEINS A, G, L IN THE AFFINITY CHROMATOGRAPHY

    OpenAIRE

    Sviatenko, О.; Gorbatiuk, O.; Vasylchenko, О.

    2014-01-01

    Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding speci...

  4. Myristylation alters DNA-binding activity and transactivation of FBR (gag-fos) protein.

    OpenAIRE

    Kamata, N; Jotte, R M; Holt, J. T.

    1991-01-01

    FBR murine sarcoma virus (gag-fos) protein, a virally transduced Fos protein, exhibits decreased gene transactivation in comparison with the cellular Fos protein. Biochemical analysis suggests that myristylation of the virally encoded N-terminal gag region results in decreased DNA binding and transcriptional activation without affecting heterodimerization with Jun protein. These findings demonstrate that protein myristylation can modulate gene regulation by a DNA-binding protein.

  5. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bojadzsieva, M.; Kocsar, L. (Orszagos Frederic Joliot-Curie Sugarbiologiai es Sugaregeszseguegyi Kutato Intezet, Budapest (Hungary)); Kremmer, T. (Orszagos Onkologiai Intezet, Budapest (Hungary))

    1985-01-01

    A micromethod was developed to determine the binding of anabolic steroids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline.

  6. Computational study of ligand binding in lipid transfer proteins: Structures, interfaces, and free energies of protein-lipid complexes

    OpenAIRE

    Fernandez Pacios, Luis; Gomez Casado, Cristina; Tordesillas Villuendas, Leticia; Palacín Gómez, Aranzazu; Sanchez-Monge Laguna De Rins, Maria Rosa; Díaz Perales, Araceli

    2012-01-01

    Plant nonspecific lipid transfer proteins (nsLTPs) bind a wide variety of lipids, which allows them to perform disparate functions. Recent reports on their multifunctionality in plant growth processes have posed new questions on the versatile binding abilities of these proteins. The lack of binding specificity has been customarily explained in qualitative terms on the basis of a supposed structural flexibility and nonspecificity of hydrophobic protein-ligand interactions. We present here a co...

  7. Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress

    International Nuclear Information System (INIS)

    Research highlights: → We performed comprehensive survey for proteins that bind to oxidized RNA. → HNRNPD and HNRNPC proteins were identified as oxidized RNA binding proteins. → Knockdown of HNRNPD/C expression caused increased sensitivity to H2O2. → Amounts of HNRNPD protein rapidly decreased when cells were exposed to H2O2. -- Abstract: Exposure of cells to oxygen radicals damage various biologically important molecules. Among the oxidized bases produced in nucleic acids, 8-oxo-7,8-dihydroguanine (8-oxoguanine) is particularly important since it causes base mispairing. To ensure accurate gene expression, organisms must have a mechanism to discriminate 8-oxoguanine-containing RNA from normal transcripts. We searched for proteins that specifically bind to 8-oxoguanine-containing RNA from human HeLa cell extracts, and the candidate proteins were identified using mass spectrometry. Among the identified candidates, splicing isoform 1 of heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) and splicing isoform C1 of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) exhibited strong abilities to bind to oxidized RNA. The amount of HNRNPD protein rapidly decreased when cells were exposed to hydrogen peroxide, an agent that enhances oxidative stress. Moreover, the suppression of HNRNPD expression by siRNA caused cells to exhibit an increased sensitivity to hydrogen peroxide. The application of siRNA against HNRNPC also caused an increase in sensitivity to hydrogen peroxide. Since no additive effect was observed with a combined addition of siRNAs for HNRNPD and HNRNPC, we concluded that the two proteins may function in the same mechanism for the accurate gene expression.

  8. Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Hayakawa, Hiroshi, E-mail: hiroshi@college.fdcnet.ac.jp [Department of Functional Bioscience and Advanced Science Research Center, Fukuoka Dental College, Fukuoka 814-0193 (Japan); Fujikane, Aya; Ito, Riyoko [Department of Functional Bioscience and Advanced Science Research Center, Fukuoka Dental College, Fukuoka 814-0193 (Japan); Matsumoto, Masaki; Nakayama, Keiichi I. [Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582 (Japan); Sekiguchi, Mutsuo [Department of Functional Bioscience and Advanced Science Research Center, Fukuoka Dental College, Fukuoka 814-0193 (Japan)

    2010-12-10

    Research highlights: {yields} We performed comprehensive survey for proteins that bind to oxidized RNA. {yields} HNRNPD and HNRNPC proteins were identified as oxidized RNA binding proteins. {yields} Knockdown of HNRNPD/C expression caused increased sensitivity to H{sub 2}O{sub 2}. {yields} Amounts of HNRNPD protein rapidly decreased when cells were exposed to H{sub 2}O{sub 2}. -- Abstract: Exposure of cells to oxygen radicals damage various biologically important molecules. Among the oxidized bases produced in nucleic acids, 8-oxo-7,8-dihydroguanine (8-oxoguanine) is particularly important since it causes base mispairing. To ensure accurate gene expression, organisms must have a mechanism to discriminate 8-oxoguanine-containing RNA from normal transcripts. We searched for proteins that specifically bind to 8-oxoguanine-containing RNA from human HeLa cell extracts, and the candidate proteins were identified using mass spectrometry. Among the identified candidates, splicing isoform 1 of heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) and splicing isoform C1 of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) exhibited strong abilities to bind to oxidized RNA. The amount of HNRNPD protein rapidly decreased when cells were exposed to hydrogen peroxide, an agent that enhances oxidative stress. Moreover, the suppression of HNRNPD expression by siRNA caused cells to exhibit an increased sensitivity to hydrogen peroxide. The application of siRNA against HNRNPC also caused an increase in sensitivity to hydrogen peroxide. Since no additive effect was observed with a combined addition of siRNAs for HNRNPD and HNRNPC, we concluded that the two proteins may function in the same mechanism for the accurate gene expression.

  9. IRBP-like proteins in the eyes of six cephalopod species--immunochemical relationship to vertebrate interstitial retinol-binding protein (IRBP) and cephalopod retinal-binding protein.

    Science.gov (United States)

    Fong, S L; Lee, P G; Ozaki, K; Hara, R; Hara, T; Bridges, C D

    1988-01-01

    SDS polyacrylamide gel electrophoresis and immunoblotting were used to examine soluble proteins from the eyes of six species of cephalopods i.e. Lolliguncula brevis, Sepia officinalis, Octopus maya, Octopus bimaculoides, Rossia pacifica and Loligo opalescens. All species had a protein ("IRBP") with molecular weight virtually identical with vertebrate interstitial retinol-binding protein (IRBP) averaging 132,400 +/- 700 (n = 6). "IRBP" reacted on nitrocellulose blot transfers with rabbit antibovine IRBP and rabbit antifrog IRBP antibodies. Unlike vertebrate IRBP, cephalopod "IRBP" (from L. brevis) did not bind exogenous retinol or concanavalin A. The N-terminal amino acid appeared to be blocked in samples electroeluted from SDS gels. The antifrog IRBP antibodies also reacted with a series of proteins with molecular weights between 46,000 and 47,000, identified as retinal-binding protein (RALBP) with anti-RALBP antibodies. Anti-IRBP also reacted with pure RALBP prepared from Todarodes pacificus. Occasionally, anti-RALBP antibodies were seen to react weakly with "IRBP" in some cephalopods. We conclude that RALBP, cephalopod "IRBP" and vertebrate IRBP share a common but distant ancestry, and that a protein resembling IRBP appeared before the vertebrates diverged from the invertebrates. Both RALBP and IRBP appear to have analogous functions in shuttling retinoids between rhodopsin and the corresponding isomerizing system, retinochrome in the cephalopods and retinol isomerase in the vertebrates. The function of cephalopod "IRBP" is unknown. PMID:3195063

  10. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins.

    Science.gov (United States)

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-04-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  11. Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein.

    OpenAIRE

    Mukherjee, A; Dai, K; Lutkenhaus, J

    1993-01-01

    FtsZ is an essential cell division protein in Escherichia coli that forms a ring structure at the division site under cell cycle control. The dynamic nature of the FtsZ ring suggests possible similarities to eukaryotic filament forming proteins such as tubulin. In this study we have determined that FtsZ is a GTP/GDP binding protein with GTPase activity. A short segment of FtsZ is homologous to a segment in tubulin believed to be involved in the interaction between tubulin and guanine nucleoti...

  12. Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

    Science.gov (United States)

    Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

    2014-06-01

    Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

  13. Isolation and characterizations of oxalate-binding proteins in the kidney

    International Nuclear Information System (INIS)

    Highlights: ► The first large-scale characterizations of oxalate-binding kidney proteins. ► The recently developed oxalate-conjugated EAH Sepharose 4B beads were applied. ► 38 forms of 26 unique oxalate-binding kidney proteins were identified. ► 25/26 (96%) of identified proteins had “L-x(3,5)-R-x(2)-[AGILPV]” domain. -- Abstract: Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) to resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed “L-x(3,5)-R-x(2)-[AGILPV]” as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone modulators.

  14. Somatomedin-1 binding protein-3: insulin-like growth factor-1 binding protein-3, insulin-like growth factor-1 carrier protein.

    Science.gov (United States)

    2003-01-01

    Somatomedin-1 binding protein-3 [insulin-like growth factor-1 binding protein-3, SomatoKine] is a recombinant complex of insulin-like growth factor-1 (rhIGF-1) and binding protein-3 (IGFBP-3), which is the major circulating somatomedin (insulin-like growth factor) binding protein; binding protein-3 regulates the delivery of somatomedin-1 to target tissues. Somatomedin-1 binding protein-3 has potential as replacement therapy for somatomedin-1 which may become depleted in indications such as major surgery, organ damage/failure and traumatic injury, resulting in catabolism. It also has potential for the treatment of osteoporosis; diseases associated with protein wasting including chronic renal failure, cachexia and severe trauma; and to attenuate cardiac dysfunction in a variety of disease states, including after severe burn trauma. Combined therapy with somatomedin-1 and somatomedin-1 binding protein-3 would prolong the duration of action of somatomedin-1 and would reduce or eliminate some of the undesirable effects associated with somatomedin-1 monotherapy. Somatomedin-1 is usually linked to binding protein-3 in the normal state of the body, and particular proteases clip them apart in response to stresses and release somatomedin-1 as needed. Therefore, somatomedin-1 binding protein-3 is a self-dosing system and SomatoKine would augment the natural supply of these linked compounds. Somatomedin-1 binding protein-3 was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on June 1 2000. Insmed and Avecia, UK, have signed an agreement for the manufacturing of SomatoKine and its components, IGF-1 and binding protein-3. CGMP clinical production of SomatoKine and its components will be done in Avecia's Advanced Biologics Centre, Billingham, UK, which manufactures recombinant-based medicines and vaccines with a capacity of up to 1000 litres. In 2003, manufacturing of SomatoKine is

  15. Calciomics:prediction and analysis of EF-hand calcium binding proteins by protein engineering

    Institute of Scientific and Technical Information of China (English)

    YANG; Jenny; Jie

    2010-01-01

    Ca2+ plays a pivotal role in the physiology and biochemistry of prokaryotic and mammalian organisms.Viruses also utilize the universal Ca2+ signal to create a specific cellular environment to achieve coexistence with the host,and to propagate.In this paper we first describe our development of a grafting approach to understand site-specific Ca2+ binding properties of EF-hand proteins with a helix-loop-helix Ca2+ binding motif,then summarize our prediction and identification of EF-hand Ca2+ binding sites on a genome-wide scale in bacteria and virus,and next report the application of the grafting approach to probe the metal binding capability of predicted EF-hand motifs within the streptococcal hemoprotein receptor(Shr) of Streptococcus pyrogenes and the nonstructural protein 1(nsP1) of Sindbis virus.When methods such as the grafting approach are developed in conjunction with prediction algorithms we are better able to probe continuous Ca2+-binding sites that have been previously underrepresented due to the limitation of conventional methodology.

  16. NRIP, a novel calmodulin binding protein, activates calcineurin to dephosphorylate human papillomavirus E2 protein.

    Science.gov (United States)

    Chang, Szu-Wei; Tsao, Yeou-Ping; Lin, Chia-Yi; Chen, Show-Li

    2011-07-01

    Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression. PMID:21543494

  17. Characterization of a small acyl-CoA-binding protein (ACBP) from Helianthus annuus L. and its binding affinities.

    Science.gov (United States)

    Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Du, Zhi-Yan; Garcés, Rafael; Tanner, Julian A; Chye, Mee-Len; Martínez-Force, Enrique; Salas, Joaquín J

    2016-05-01

    Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered. PMID:26938582

  18. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    Directory of Open Access Journals (Sweden)

    Yu Zhu

    2015-11-01

    Full Text Available Chitin-binding proteins (CBPs are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton.

  19. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin.

    Science.gov (United States)

    Zhu, Yu; Ye, Xin-Hai; Liu, Yang; Yan, Zhi-Chao; Stanley, David; Ye, Gong-Yin; Fang, Qi

    2015-12-01

    Chitin-binding proteins (CBPs) are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP) from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs) of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton. PMID:26633500

  20. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    Science.gov (United States)

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-01

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites. PMID:27015007

  1. Binding of cationic surfactants to DNA, protein and DNA-protein mixtures.

    Science.gov (United States)

    Gani, S A; Chattoraj, D K; Mukherjee, D C

    1999-06-01

    Extent of binding (gamma 2(1)) of cationic surfactants cetyltrimethyl ammonium bromide (CTAB), myristyltrimethyl ammonium bromide (MTAB) and dodecyl trimethyl ammonium bromide (DTAB) to calf-thymus DNA, bovine serum albumin (BSA) and to their binary mixture respectively have been measured as function of bulk concentration of the surfactant by using equilibrium dialysis technique. Binding of CTAB has been studied at different pH, ionic strength (mu), temperature and biopolymer composition and with native and denatured states of the biopolymers. The chain-length of different long chain amines plays a significant role in the extent of binding under identical solution condition. The binding ratios for CTAB to collagen, gelatin, DNA-collagen and DNA-gelatin mixtures respectively have also been determined. The conformational structures of different biopolymers are observed to play significant role in macromolecular interactions between protein and DNA in the presence of CTAB. From the experimental values of the maximum binding ratio (gamma 2m) at the saturation level for each individual biopolymer, ideal values (gamma 2m)id have been theoretically calculated for binary mixtures of biopolymers using additivity rule. The protein-DNA-CTAB interaction in mixture has been explained in terms of the deviation (delta) of (gamma 2m) from (gamma 2m)id in the presence of a surfactant in bulk. The binding of surfactants to biopolymers and to their binary mixtures are compared more precisely in terms of the Gibbs' free energy decrease (-delta G degree) for the saturation of the binding sites in the biopolymers or biopolymer mixtures with the change of the bulk surfactant activity from zero to unity in the rational mole fraction scale. PMID:10650715

  2. Regulation of insulin-like growth factor binding protein-1 (IGFBP-1) and implications in catabolic conditions

    OpenAIRE

    Lindgren, Björn

    1997-01-01

    This thesis has studied the regulation of IGFBP-1 (insulin-like growth factor binding protein 1), which is one factor regulating the bioavailability of IGF-I with special interest how IGFBP-1 is regulated in vitro and in humans, especially in diabetes and catabolic conditions. The IGFBP-1 cDNA was cloned and used for studies in human hepatoma cells, HepG2, which showed that both insulin and IGF-I could decrease IGFBP-1 in the cell conditioned medium. IGF-I inhibited also IGF...

  3. Novel RNA-binding properties of the MTG chromatin regulatory proteins

    Directory of Open Access Journals (Sweden)

    Sacchi Nicoletta

    2008-10-01

    Full Text Available Abstract Background The myeloid translocation gene (MTG proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the DNA-binding domain of AML1, a transcriptional activator crucial for hematopoiesis. The AML1-MTG fusion proteins, as the wild type MTGs, display four conserved homology regions (NHR1-4 related to the Drosophila nervy protein. Structural protein analyses led us to test the hypothesis that specific MTG domains may mediate RNA binding. Results By using an RNA-binding assay based on synthetic RNA homopolymers and a panel of MTG deletion mutants, here we show that all the MTG proteins can bind RNA. The RNA-binding properties can be traced to two regions: the Zinc finger domains in the NHR4, which mediate Zinc-dependent RNA binding, and a novel short basic region (SBR upstream of the NHR2, which mediates Zinc-independent RNA binding. The two AML1-MTG fusion proteins, retaining both the Zinc fingers domains and the SBR, also display RNA-binding properties. Conclusion Evidence has been accumulating that RNA plays a role in transcriptional control. Both wild type MTGs and chimeric AML1-MTG proteins display in vitro RNA-binding properties, thus opening new perspectives on the possible involvement of an RNA component in MTG-mediated chromatin regulation.

  4. Protein-ligand binding affinities from large-scale quantum mechanical simulations

    OpenAIRE

    Fox, Stephen J.

    2012-01-01

    The accurate prediction of protein-drug binding affinities is a major aim of computational drug optimisation and development. A quantitative measure of binding affinity is provided by the free energy of binding, and such calculations typically require extensive configurational sampling of entities such as proteins with thousands of atoms. Current binding free energy methods use force fields to perform the configurational sampling and to compute interaction energies. Due to the empirical natur...

  5. Protein and solvent dynamics of the water-soluble chlorophyll-binding protein (WSCP)

    International Nuclear Information System (INIS)

    This study presents quasielastic neutron scattering data of the water-soluble chlorophyll-binding protein (WSCP) and the corresponding buffer solution at room temperature. The contributions of protein and buffer solution to the overall scattering are carefully separated. Otherwise, the fast water dynamics dominating the buffer contribution is likely to mask the slow protein dynamics. In the case of WSCP, the protein scattering can be described by two contributions: first, internal protein dynamics represented by a diffusion in a sphere with an average radius of 2.7 Angstroms and secondly global (Brownian) diffusion of the WSCP macromolecule with an upper limit for the translational diffusion coefficient of 9.4*10-7 cm2/s. (authors)

  6. Characterization of the retinoblastoma binding proteins RBP1 and RBP2

    DEFF Research Database (Denmark)

    Fattaey, A R; Helin, K; Dembski, M S;

    1993-01-01

    The retinoblastoma gene product, pRB, regulates cell proliferation by binding to and inhibiting the activity of key growth promoting proteins. Several cellular proteins have been shown to bind directly to pRB and the genes encoding a number of them have been isolated. The protein product of one of...

  7. Inhibition of the vitamin B12 binding capacity of proteins by the hydrolysis product of cyclophosphamide

    International Nuclear Information System (INIS)

    The inhibitory effect of cyclophosphamide hydrolysis product (CPHP) on vitamin B12 binding ability to proteins has been established. The ester N-(2-chloroethyl)-N'-(3-phosphopropyl)-etheylenediamine hydrochloride is probably responsible, in vitro, for blocking the protein binding sites. Preincubation of proteins with vitamin B12 prevents the inhibitory effect of CPHP. (au)

  8. Glycosylation status of vitamin D binding protein in cancer patients.

    Science.gov (United States)

    Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

    2009-10-01

    On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages. PMID:19642159

  9. Convolutional neural network architectures for predicting DNA–protein binding

    Science.gov (United States)

    Zeng, Haoyang; Edwards, Matthew D.; Liu, Ge; Gifford, David K.

    2016-01-01

    Motivation: Convolutional neural networks (CNN) have outperformed conventional methods in modeling the sequence specificity of DNA–protein binding. Yet inappropriate CNN architectures can yield poorer performance than simpler models. Thus an in-depth understanding of how to match CNN architecture to a given task is needed to fully harness the power of CNNs for computational biology applications. Results: We present a systematic exploration of CNN architectures for predicting DNA sequence binding using a large compendium of transcription factor datasets. We identify the best-performing architectures by varying CNN width, depth and pooling designs. We find that adding convolutional kernels to a network is important for motif-based tasks. We show the benefits of CNNs in learning rich higher-order sequence features, such as secondary motifs and local sequence context, by comparing network performance on multiple modeling tasks ranging in difficulty. We also demonstrate how careful construction of sequence benchmark datasets, using approaches that control potentially confounding effects like positional or motif strength bias, is critical in making fair comparisons between competing methods. We explore how to establish the sufficiency of training data for these learning tasks, and we have created a flexible cloud-based framework that permits the rapid exploration of alternative neural network architectures for problems in computational biology. Availability and Implementation: All the models analyzed are available at http://cnn.csail.mit.edu. Contact: gifford@mit.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307608

  10. Heterogeneity of binding subunits of the human 150K insulin-like growth factor binding protein.

    Science.gov (United States)

    Gelato, M C; Gaynes, L A; Greenstein, L A; Nissley, S P

    1990-04-01

    Models for the structure of the GH-dependent 150K insulin-like growth factor-binding protein (IGF-BP) complex include 1) a binding subunit of 40-60K mol wt associated with a larger nonbinding component, and 2) an oligomeric structure simply made up of six 25-28K monomeric IGF-BP complexes. To evaluate these alternative models we examined the IGF-binding characteristics and behavior on an SP-Sephadex ion exchange column of BP species identified by chemically cross-linking [125I]IGF-I and [125I]IGF-II. In addition, human serum was gel filtered on Sephadex G-200 in 0.05 M NH4HCO3, pH 8.0, and the 150K BP identified by binding of [125I]IGF-II to column fractions. When [125I]IGF-I or [125I]IGF-II was cross-linked to the 150K BP with disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10-15%) and autoradiography, four specifically labeled complexes of 20K, 24K, 33K, and 47K mol wt were identified. We examined the IGF-binding characteristics of these species by cross-linking [125I]IGF-I and [125I]IGF-II after incubation in the presence of increasing concentrations of unlabeled IGF-I or IGF-II. Formation of the 24K complex was inhibited more potently by IGF-II than IGF-I, whereas the relative potency of IGF-I vs. IGF-II for inhibition of the formation of the other complexes depended upon whether [125I]IGF-II or [125I]IGF-I was used. When the 150K BP complex generated from gel filtration on Sephadex G-200 was acid stripped, the only species seen with chemical cross-linking of either [125I]IGF-I or [125I]IGF-II was the 47K complex. By both conventional competitive binding studies and cross-linking [125I]IGF-I and [125I]IGF-II after incubation with increasing concentrations of unlabeled IGF-I or IGF-II, the formation of the 47K complex was usually more potently inhibited by IGF-I than IGF-II. When Cohn fraction IV extract was chromatographed on a SP-Sephadex column (pH 3) and cross-linking performed on the flow-through, the 47K

  11. Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins.

    Science.gov (United States)

    Wang, Jilite; Shimada, Masaya; Kato, Yukina; Kusada, Mio; Nagaoka, Satoshi

    2015-01-01

    Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins. PMID:25374002

  12. A Column Free Protein Purification Procedure using E. coli Single ‐ Stranded DNA Binding Protein (SSB) as an Affinity Tag

    OpenAIRE

    Soffe, Mark

    2014-01-01

    SSBs are DNA binding proteins that are essential components of cells and play key roles in DNA replication, repair, and recombination. Here we utilize two biochemical properties associated with the E. coli SSB protein to develop a novel procedure to purify proteins using a resin-free strategy to combat the largest bottleneck in biochemical research— obtaining uncontaminated, single proteins from the total cellular contents. 1. E. coli SSB binds to single stranded DNA (ssDNA) with extremely...

  13. Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein

    OpenAIRE

    Shen, Wen-Jun; Sridhar, Kunju; Bernlohr, David A.; Fredric B Kraemer

    1999-01-01

    Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that binds fatty acids, retinoids, and other hydro...

  14. A conformational analysis of Walker motif A [GXXXXGKT (S)] in nucleotide-binding and other proteins

    OpenAIRE

    Ramakrishnan, C; Dani, VS; Ramasarma, T

    2002-01-01

    The sequence GXXXXGKT/S, popularly known as Walker motif A, is widely believed to be the site for binding nucleotides in many proteins. Examination of the crystal structures in the Protein Data Bank showed that about half of the examples having these sequences do not bind or use nucleotides. Data analyses showed 92 different Walker sequences of the variable quartet (XXXX). Ramachandran angles in this segment revealed conformational similarity in the group of 45 proteins, known to bind or util...

  15. Isothermal Chemical Denaturation to Determine Binding Affinity of Small Molecules to G-Protein Coupled Receptors

    OpenAIRE

    Ross, Patrick; Weihofen, Wilhelm; Siu, Fai; Xie, Amy; Katakia, Hetal; Wright, S. Kirk; Hunt, Ian; Brown, Richard K; Freire, Ernesto

    2014-01-01

    The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation (ICD) has been shown to be a valuable biophysical method to determine in a direct and label-free fashion the binding of ligands to soluble proteins. In this communication, the application of isothermal che...

  16. Isotope-coded ATP Probe for Quantitative Affinity Profiling of ATP-binding Proteins

    OpenAIRE

    Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

    2013-01-01

    ATP-binding proteins play significant roles in numerous cellular processes. Here, we introduced a novel isotope-coded ATP-affinity probe (ICAP) as acylating agent to simultaneously enrich and incorporate isotope label to ATP-binding proteins. By taking advantage of the quantitative capability of this isotope-coded probe, we devised an affinity profiling strategy to comprehensively characterize ATP-protein interactions at the entire proteome scale. False-positive identification of ATP-binding ...

  17. The effects of GH and hormone replacement therapy on serum concentrations of mannan-binding lectin, surfactant protein D and vitamin D binding protein in Turner syndrome

    DEFF Research Database (Denmark)

    Gravholt, Claus Højbjerg; Leth-Larsen, Rikke; Lauridsen, Anna Lis;

    2004-01-01

    function. In the present study we examined whether GH or hormone replacement therapy (HRT) in Turner syndrome (TS) influence the serum concentrations of MBL and two other proteins partaking in the innate immune defence, surfactant protein D (SP-D) and vitamin D binding protein (DBP). DESIGN: Study 1: a...

  18. Split green fluorescent protein as a modular binding partner for protein crystallization

    International Nuclear Information System (INIS)

    A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization

  19. Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata

    Czech Academy of Sciences Publication Activity Database

    Siciliano, P.; He, X. L.; Woodcock, C.; Pickett, J. A.; Field, L. M.; Birkett, M. A.; Kalinová, Blanka; Gomulski, L. M.; Scolari, F.; Gasperi, G.; Malacrida, A. R.; Zhou, J. J.

    2014-01-01

    Roč. 48, May (2014), s. 51-62. ISSN 0965-1748 Institutional support: RVO:61388963 Keywords : medfly * Ceratitis capitata * olfaction * odorant binding protein * pheromone binding protein * pheromone * binding studies * protein expression * electroantennography * GC-EAG * fluorescence displacement Subject RIV: CE - Biochemistry Impact factor: 3.450, year: 2014

  20. Acute hantavirus infection induces galectin-3-binding protein.

    Science.gov (United States)

    Hepojoki, Jussi; Strandin, Tomas; Hetzel, Udo; Sironen, Tarja; Klingström, Jonas; Sane, Jussi; Mäkelä, Satu; Mustonen, Jukka; Meri, Seppo; Lundkvist, Ake; Vapalahti, Olli; Lankinen, Hilkka; Vaheri, Antti

    2014-11-01

    Hantaviruses are zoonotic viruses that cause life-threatening diseases when transmitted to humans. Severe hantavirus infection is manifested by impairment of renal function, pulmonary oedema and capillary leakage. Both innate and adaptive immune responses contribute to the pathogenesis, but the underlying mechanisms are not fully understood. Here, we showed that galectin-3-binding protein (Gal-3BP) was upregulated as a result of hantavirus infection both in vitro and in vivo. Gal-3BP is a secreted glycoprotein found in human serum, and increased Gal-3BP levels have been reported in chronic viral infections and in several types of cancer. Our in vitro experiments showed that, whilst Vero E6 cells (an African green monkey kidney cell line) constitutively expressed and secreted Gal-3BP, this protein was detected in primary human cells only as a result of hantavirus infection. Analysis of Gal-3BP levels in serum samples of cynomolgus macaques infected experimentally with hantavirus indicated that hantavirus infection induced Gal-3BP also in vivo. Finally, analysis of plasma samples collected from patients hospitalized because of acute hantavirus infection showed higher Gal-3BP levels during the acute than the convalescent phase. Furthermore, the Gal-3BP levels in patients with haemorrhagic fever with renal syndrome correlated with increased complement activation and with clinical variables reflecting the severity of acute hantavirus infection. PMID:25013204

  1. QM/MM Molecular Dynamics Studies of Metal Binding Proteins

    Directory of Open Access Journals (Sweden)

    Pietro Vidossich

    2014-07-01

    Full Text Available Mixed quantum-classical (quantum mechanical/molecular mechanical (QM/MM simulations have strongly contributed to providing insights into the understanding of several structural and mechanistic aspects of biological molecules. They played a particularly important role in metal binding proteins, where the electronic effects of transition metals have to be explicitly taken into account for the correct representation of the underlying biochemical process. In this review, after a brief description of the basic concepts of the QM/MM method, we provide an overview of its capabilities using selected examples taken from our work. Specifically, we will focus on heme peroxidases, metallo-β-lactamases, α-synuclein and ligase ribozymes to show how this approach is capable of describing the catalytic and/or structural role played by transition (Fe, Zn or Cu and main group (Mg metals. Applications will reveal how metal ions influence the formation and reduction of high redox intermediates in catalytic cycles and enhance drug metabolism, amyloidogenic aggregate formation and nucleic acid synthesis. In turn, it will become manifest that the protein frame directs and modulates the properties and reactivity of the metal ions.

  2. Serum protein inhibition of thyrotropin binding to human thyroid tissue

    International Nuclear Information System (INIS)

    We used a modificaton of the TSH radioreceptor assay to detect TSH-binding inhibition (TBI) activity in serum and serum fractions from normal subjects and patients with Graves' disease. TBI activity is present in normal IgG prepared by DEAE-Sephadex chromatography and in normal globulins prepared by precipitation at 1.6 M ammonium sulfate. Other normal serum proteins also had TBI activity when large concentrations were tested. Gel filtration chromatography and powder block electrophoresis were used to prepare fractions of normal and Graves' disease sera. In these fractions from normal serum, TBI activity was found in both γ-globulin and α-globulin-albumin fractions electrophoretically and in both 7S and 4S peaks from gel filtration. TBI activity from Graves' disease patients' sera was similarly distributed, but relatively more TBI accompanied the electrophoretic γ-globulins. Sepharose Protein-A and anti-IgG were used as immunoabsorbents to isolate and purify IgG from normal and Graves' disease sera. TBI activity in IgG was proportional to the IgG concentration, indicating that the TBI which migrates as a γ-globulin electrophoretically is an IgG and thus may possibly be an antibody. Inhibitory activity found in normal serum globulins and in the non-IgG fractions of both normal and abnormal sera seriously interferes with attempts to use the TSH radioreceptor assay to study the hypothesized anti-TSH receptor antibody in the serum of patients with Graves' disease

  3. Is vitamin D binding protein a novel predictor of labour?

    Directory of Open Access Journals (Sweden)

    Stella Liong

    Full Text Available Vitamin D binding protein (VDBP has previously been identified in the amniotic fluid and cervicovaginal fluid (CVF of pregnant women. The biological functions of VDBP include acting as a carrier protein for vitamin D metabolites, the clearance of actin that is released during tissue injury and the augmentation of the pro-inflammatory response. This longitudinal observational study was conducted on 221 healthy pregnant women who spontaneously laboured and delivered either at term or preterm. Serial CVF samples were collected and VDBP was measured by ELISA. Binary logistic regression analysis was performed to assess the utility of VDBP as a predictor of labour. VDBP in the CVF did not change between 20 and 35 weeks' gestation. VDBP measured in-labour was significantly increased 4.2 to 7.4-fold compared to 4-7, 8-14 and 15-28 days before labour (P<0.05. VDBP concentration was 4.3-fold significantly higher at 0-3 days compared to 15-28 days pre-labour (P<0.05. The efficacy of VDBP to predict spontaneous labour onset within 3 days provided a positive and negative predictive value of 82.8% and 95.3% respectively (area under receiver operator characteristic curve  = 0.974. This longitudinal study of pregnant women suggests that VDBP in the CVF may be a useful predictor of labour.

  4. Suppression of cellular transformation by poly (A binding protein interacting protein 2 (Paip2.

    Directory of Open Access Journals (Sweden)

    Amy B Rosenfeld

    Full Text Available Controlling translation is crucial for the homeostasis of a cell. Its deregulation can facilitate the development and progression of many diseases including cancer. Poly (A binding protein interacting protein 2 (Paip2 inhibits efficient initiation of translation by impairing formation of the necessary closed loop of mRNA. The over production of Paip2 in the presence of a constitutively active form of hRas(V12 can reduce colony formation in a semi-solid matrix and focus formation on a cell monolayer. The ability of Paip2 to bind to Pabp is required to suppress the transformed phenotype mediated by hRas(V12. These observations indicate that Paip2 is able to function as a tumor suppressor.

  5. Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C

    Directory of Open Access Journals (Sweden)

    Riedemann Johann

    2011-05-01

    Full Text Available Abstract Background Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA. Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs. Cardiac Myosin Binding Protein-C (cMyBPC and cardiac troponin I (cTNI are hypertrophic cardiomyopathy (HCM-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation. Results During a yeast 2-hybrid (Y2H library screen using a trisphosphorylation mimic of the C1-C2 region of cMyBPC, we identified isoform 4 of myomegalin (MMGL as an interactor of this N-terminal cMyBPC region. As MMGL has previously been shown to interact with phosphodiesterase 4D, we speculated that it may be a PKA-anchoring protein (AKAP. To investigate this possibility, we assessed the ability of MMGL isoform 4 to interact with PKA regulatory subunits R1A and R2A using Y2H-based direct protein-protein interaction assays. Additionally, to further elucidate the function of MMGL, we used it as bait to screen a cardiac cDNA library. Other PKA targets, viz. CARP, COMMD4, ENO1, ENO3 and cTNI were identified as putative interactors, with cTNI being the most frequent interactor. We further assessed and confirmed these interactions by fluorescent 3D-co-localization in differentiated H9C2 cells as well as by in vivo co-immunoprecipitation. We also showed that quantitatively more interaction occurs between MMGL and cTNI under β-adrenergic stress. Moreover, siRNA-mediated knockdown of MMGL leads to reduction of cMyBPC levels under conditions of adrenergic stress, indicating that MMGL-assisted phosphorylation is requisite for protection of cMyBPC against proteolytic cleavage. Conclusions This study ascribes a novel function to MMGL isoform 4: it meets all criteria for classification as an AKAP, and we show that is involved in the phosphorylation of cMyBPC as well as cTNI, hence MMGL

  6. Penicillin binding proteins as danger signals: meningococcal penicillin binding protein 2 activates dendritic cells through Toll-like receptor 4.

    Directory of Open Access Journals (Sweden)

    Marcelo Hill

    Full Text Available Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml ± 0.1, CD80 (LOGEC50 = 4.88 µg/ml ± 0.15 and CD86 (LOGEC50 = 5.36 µg/ml ± 0.1. This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7 ± 5.1% cells versus 12 ± 2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed thatPBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.

  7. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    Science.gov (United States)

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research. PMID:16302727

  8. Significance of lipopolysaccharide-binding protein (an acute phase protein) in monitoring critically ill patients

    OpenAIRE

    Prucha, Miroslav; Herold, Ivan; Zazula, Roman; Dubska, Ladislava; Dostal, Miroslav; Hildebrand, Thomas; Hyanek, Josef

    2003-01-01

    Introduction The present study was conducted to assess the value of serum concentration of lipopolysaccharide-binding protein (LBP) in patients with systemic inflammatory response syndrome (SIRS), sepsis and septic shock with respect to its ability to differentiate between infectious and noninfectious etiologies in SIRS and to predict prognosis. Methods This prospective cohort study was conducted in a multidisciplinary intensive care unit. Sixty-eight patients, admitted consecutively to the i...

  9. Identification of Pneumococcal Surface Protein A as a Lactoferrin-Binding Protein of Streptococcus pneumoniae

    OpenAIRE

    Hammerschmidt, Sven; Bethe, Gesina; H. Remane, Petra; Chhatwal, Gursharan S.

    1999-01-01

    Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10−18 M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that Streptococcus pneumoniae specifically recognizes and binds the iron carrier protein human Lf (hLf). Pretreatment of pneumococci with proteases reduced hL...

  10. Biointerface: protein enhanced stem cells binding to implant surface.

    Science.gov (United States)

    Chrzanowski, W; Kondyurin, A; Lee, Jae Ho; Lord, Megan S; Bilek, M M M; Kim, Hae-Won

    2012-09-01

    The number of metallic implantable devices placed every year is estimated at 3.7 million. This number has been steadily increasing over last decades at a rate of around 8 %. In spite of the many successes of the devices the implantation of biomaterial into tissues almost universally leads to the development of an avascular sac, which consists of fibrous tissue around the device and walls off the implant from the body. This reaction can be detrimental to the function of implant, reduces its lifetime, and necessitates repeated surgery. Clearly, to reduce the number of revision surgeries and improve long-term implant function it is necessary to enhance device integration by modulating cell adhesion and function. In this paper we have demonstrated that it is possible to enhance stem cell attachment using engineered biointerfaces. To create this functional interface, samples were coated with polymer (as a precursor) and then ion implanted to create a reactive interface that aids the binding of biomolecules--fibronectin. Both AFM and XPS analyses confirmed the presence of protein layers on the samples. The amount of protein was significant greater for the ion implanted surfaces and was not disrupted upon washing with detergent, hence the formation of strong bonds with the interface was confirmed. While, for non ion implanted surfaces, a decrease of protein was observed after washing with detergent. Finally, the number of stem cells attached to the surface was enhanced for ion implanted surfaces. The studies presented confirm that the developed bionterface with immobilised fibronectin is an effective means to modulate stem cell attachment. PMID:22714559

  11. Regulation of RNA binding proteins in trypanosomatid protozoan parasites.

    Science.gov (United States)

    Romaniuk, María Albertina; Cervini, Gabriela; Cassola, Alejandro

    2016-02-26

    Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3' untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy. PMID:26981203

  12. Changes in GDP binding to brown adipose tissue mitochondria and the uncoupling protein

    International Nuclear Information System (INIS)

    Incubation in vitro of brown adipose tissue (BAT) mitochondria with divalent cations, spermine, or alkaline phosphatase led to a marked increase in the binding of [3H]GDP. The effect of Mg2+ appeared to be the most specific and led to the largest increase in GDP binding. A simplified method was developed for measuring GDP binding to purified uncoupling protein from rat BAT mitochondria. Application of this method indicates that uncoupling protein from cold-acclimated rats binds twice as much GDP as uncoupling protein from cold-acclimated rats that were briefly returned to thermoneutrality, paralleling changes in GDP binding to the mitochondria. Incubation of BAT mitochondria with Mg2+ led to a smaller increase in GDP binding to the subsequently purified uncoupling protein, suggesting that divalent cations may somehow participate in the regulation of the activity of the uncoupling protein

  13. Characterization of a zinc blotting technique: evidence that a retroviral gag protein binds zinc

    International Nuclear Information System (INIS)

    We have characterized a simple method that uses 65ZnCl2 to detect zinc-binding proteins that have been immobilized on nitrocellulose. Conditions have been identified that permit the detection of as little as 1 microgram of some zinc-binding proteins. The specificity of the binding is indicated by the ability of other divalent metal ions to compete with 65Zn(II) in this assay. We have used this technique to provide evidence that the nucleic acid-binding gag protein of retroviruses also binds zinc. This technique can be applied to biological mixtures of proteins and may be used in proteolytic mapping studies to identify protein fragments that have zinc-binding activity

  14. Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

    Directory of Open Access Journals (Sweden)

    TIJANA SAVIC

    2006-02-01

    Full Text Available In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

  15. Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

    International Nuclear Information System (INIS)

    A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions

  16. Detection and properties of A-factor-binding protein from Streptomyces griseus

    International Nuclear Information System (INIS)

    The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The inducing material virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein

  17. Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

    Science.gov (United States)

    DeVree, Brian T; Mahoney, Jacob P; Vélez-Ruiz, Gisselle A; Rasmussen, Soren G F; Kuszak, Adam J; Edwald, Elin; Fung, Juan-Jose; Manglik, Aashish; Masureel, Matthieu; Du, Yang; Matt, Rachel A; Pardon, Els; Steyaert, Jan; Kobilka, Brian K; Sunahara, Roger K

    2016-07-01

    G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity. PMID:27362234

  18. Antibody Responses in Patients with Staphylococcal Septicemia against Two Staphylococcus aureus Fibrinogen Binding Proteins: Clumping Factor and an Extracellular Fibrinogen Binding Protein

    OpenAIRE

    Colque-Navarro, Patricia; Palma, Marco; Söderquist, Bo; Flock, Jan-Ingmar; Möllby, Roland

    2000-01-01

    We analyzed the serum antibody responses against two Staphylococcus aureus fibrinogen binding proteins, the cell-bound clumping factor (Clf) and an extracellular fibrinogen binding protein (Efb). The material consisted of 105 consecutive serum samples from 41 patients suffering from S. aureus septicemia and 72 serum samples from healthy individuals. An enzyme-linked immunosorbent assay (ELISA) was developed. Healthy individuals showed variable levels of antibodies against the studied antigens...

  19. Secretion and Proteolysis of Heterologous Proteins Fused to the Escherichia coli Maltose Binding Protein in Pichia pastoris

    OpenAIRE

    Li, Zhiguo; Leung, Wilson; Yon, Amy; Nguyen, John; Perez, Vincent C.; Vu, Jane; Giang, William; Luong, Linda T.; Phan, Tracy; Salazar, Katherine A.; Gomez, Seth R.; Au, Colin; Xiang, Fan; Thomas, David W; Franz, Andreas H.

    2010-01-01

    The E. coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N-terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris, a popular eukaryotic host for heterologous protein expression. Whe...

  20. Standardization for cortisol determination in human blood by competitive protein-binding

    International Nuclear Information System (INIS)

    Standardization for determination of cortisol from human plasma (17-hydroxycorticosteroids) using competitive protein-binding method is presented. Activated carbon coated with dextrans is used for separation of the hormone-protein complexe and hormone labelled free

  1. The presence of phosphate-binding protein in inner mitochondrial membrane

    Directory of Open Access Journals (Sweden)

    Hatase,Osamu

    1976-06-01

    Full Text Available Phosphate-binding protein(s was found in the inner mitochondrial membrane of calf heart by Sephadex G-200 and G-25 gel filtration. The binding activity was inhibited by N-ethylmaleimide and competed by a large amount of cold phosphate. The amount of phosphate bound to the fraction was 29 nmoles per mg of protein. Affinity chromatography with phosphate-bound Sepharose 4B confirmed the presence of phosphate-binding protein(s in the active fraction of mitochondrial membrane fractionated by gel filtration.

  2. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    International Nuclear Information System (INIS)

    Highlights: ► We designed novel recombinant albumin-RBP fusion proteins. ► Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). ► Fusion proteins are successfully internalized into and inactivate PSCs. ► RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumindomainIII (R-III) and albumindomainI-RBP-albuminIII (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti

  3. Squid rhodopsin and GTP-binding protein crossreact with vertebrate photoreceptor enzymes.

    OpenAIRE

    Saibil, H R; Michel-Villaz, M

    1984-01-01

    The activation of photoreceptor GTP-binding protein by rhodopsin was studied in squid photoreceptors and in crossreactions between the squid and bovine proteins. Turbidity changes were observed in the far-red after photoexcitation of rhodopsin with brief flashes and were used to probe interactions between photoreceptor membrane suspensions and soluble protein extracts. Our findings are squid photoreceptors contain a GTP-binding protein detectable by light- and GTP-sensitive turbidity changes ...

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

    OpenAIRE

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

    2016-01-01

    Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variet...

  5. Engineering of binding affinity at metal ion binding sites for the stabilization of proteins: Subtilisin as a test case

    International Nuclear Information System (INIS)

    A weak Ca2+ binding site in the bacterial serine protease subtilisin BPN' was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca2+ binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN' at 65/degrees/C as a function of the free [Ca2+]. Increasing the [Ca2+] in the range of 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca2+ specific binding site with an apparent log K/sub a/ = 1.49. A series of refined X-ray crystal structures of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl2 has allowed a detailed structural characterization of this Ca2+ binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca2+ by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca2+ binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein. X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 /Angstrom/, respectively, from the bound Ca2+. These distances and the degree of enhanced binding are consistent with simple electrostatic theory. Moreover, when both Asp changes were introduced together, the binding affinity for Ca2+ was found to be increased about 6-fold over that for the wild-type protein, suggesting an independent and nearly additive effect on the total electrostatic potential at this locus

  6. A1C Test

    Science.gov (United States)

    ... to minimize the complications caused by chronically elevated glucose levels, such as progressive damage to body organs like the kidneys, eyes, cardiovascular system, and nerves. The A1c test result ...

  7. Detection and characterization of heparin-binding proteins with a gel overlay procedure

    International Nuclear Information System (INIS)

    The binding of 125I-labeled derivatives of heparin has been used by several investigators to identify heparin-binding fragments of different heparin-binding proteins. In this report we utilize the procedure described by J.W. Smith and D.J. Knauer (1987, Anal. Biochem. 160, 105-114) to produce 125I-fluorescein-heparin. Using this derivative, we compare the use of gel overlay procedures with Western blot procedures for the detection of heparin-binding proteins following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. We show that the gel overlay procedure is a relatively simple and sensitive method for visualizing heparin-binding proteins. In addition, we use the procedure to characterize the heparin-binding properties of heparin-binding growth factor 1 (acidic fibroblast growth factor) with synthetic peptide competitors and site-directed mutants of the growth factor

  8. Competitive protein binding analysis for thyroxine using Sephadex column (Tetralute)

    International Nuclear Information System (INIS)

    The method of competitive protein binding analysis of thyroxine (T4) using Tetralute kit was evaluated. The net retention was decreased when the procedure of competition and separation was performed at a higher temperature but the final T4-I values were constant when the standard and test sera were treated identically. Coefficient of variation (C.V.) was 4% (within-assay) and 6% (between-assay) respectively. However, the T4-I values of pooled serum for quality control were slightly lower in earlier experiments in which correction factors (1.03--1.62 in 18 out of 21 assays) were necessary. T4-I values were determined by the Tetralute in 155 cases. They were as follows: 4.9+-0.8 μg/dl (euthyroid subjects), 6.4+-1.2 μg/dl (cord serum), 7.1+-1.1 μg/dl (pregnant women). 9.0+-3.6 μg/dl (trophoblastic disease), 13.3+-4.8 μg/dl (Graves' disease), 6.3+-1.6 μg/dl (Plummer's disease), 4-I values determined by Tetralute and Res-O-Mat T4 (r=0.96). Following oral administration of Telepaque the serum protein-bound iodine was markedly elevated, while the T4-I determined by Tetralute did not change. In vitro addition of diphenylhydantoin (500 μg/ml), salicylate (4 mg/ml) and phenobarbital (1 mg/ml) had no or little effect on T4 determination by Tetralute. A high concentration of benzbromarone (0.1 mg/ml) caused a higher value of T4-I determined by Tetralute when added to a TBG solution but there was only a slight increase when it was added to serum. (auth.)

  9. Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe

    OpenAIRE

    Jun Adachi; Marina Kishida; Shio Watanabe; Yuuki Hashimoto; Kazuna Fukamizu; Takeshi Tomonaga

    2015-01-01

    Interactions between ATP and ATP-binding proteins (ATPome) are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes) that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014) [1]. As a result, we identified ...

  10. How Similar Are Protein Folding and Protein Binding Nuclei? Examination of Vibrational Motions of Energy Hot Spots and Conserved Residues

    OpenAIRE

    Haliloglu, Turkan; Keskin, Ozlem; Ma, Buyong; Nussinov, Ruth

    2004-01-01

    The underlying physico-chemical principles of the interactions between domains in protein folding are similar to those between protein molecules in binding. Here we show that conserved residues and experimental hot spots at intermolecular binding interfaces overlap residues that vibrate with high frequencies. Similarly, conserved residues and hot spots are found in protein cores and are also observed to vibrate with high frequencies. In both cases, these residues contribute significantly to t...

  11. Mechanism of Fluorescence and Conformational Changes of the Sarcoplasmic Calcium Binding Protein of the Sand Worm Nereis diversicolor upon Ca2+ or Mg2+ Binding

    OpenAIRE

    Sillen, Alain; Verheyden, Stefan; Delfosse, Lotte; Braem, Tania; Robben, Johan; Volckaert, Guido; Engelborghs, Yves

    2003-01-01

    The calcium-binding protein isolated from the sarcoplasm of the muscles of the sand worm Nereis diversicolor has four EF-hands and three active binding sites for Ca2+ or Mg2+. Nereis diversicolor sarcoplasmic calcium-binding protein contains three tryptophan residues at positions 4, 57, and 170, respectively. The Wt protein shows a very limited fluorescence increase upon binding of Ca2+ or Mg2+. Single-tryptophan-containing mutants were produced and purified. The fluorescence titrations of th...

  12. Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains.

    OpenAIRE

    Vallee, B L; Coleman, J E; Auld, D S

    1991-01-01

    We now recognize three distinct motifs of DNA-binding zinc proteins: (i) zinc fingers, (ii) zinc clusters, and (iii) zinc twists. Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses of DNA-binding zinc proteins have not been available to serve as standards of reference for the zinc binding sites of these families of proteins. Those of the DNA-binding domains of the fungal transcription factor GAL4 and the rat glucocorticoid receptor are the first to have be...

  13. Mycobacterial PE_PGRS Proteins Contain Calcium-Binding Motifs with Parallel β-roll Folds

    Institute of Scientific and Technical Information of China (English)

    Nandita; Bachhawat; Balvinder; Singh

    2007-01-01

    The PE_PGRS family of proteins unique to mycobacteria is demonstrated to con- rain multiple calcium-binding and glycine-rich sequence motifs GGXGXD/NXUX. This sequence repeat constitutes a calcium-binding parallel/3-roll or parallel β-helix structure and is found in RTX toxins secreted by many Gram-negative bacteria. It is predicted that the highly homologous PE_PGRS proteins containing multiple copies of the nona-peptide motif could fold into similar calcium-binding structures. The implication of the predicted calcium-binding property of PE_PGRS proteins in the Ught of macrophage-pathogen interaction and pathogenesis is presented.

  14. OB protein binds specifically to the choroid plexus of mice and rats.

    Science.gov (United States)

    Devos, R; Richards, J G; Campfield, L A; Tartaglia, L A; Guisez, Y; van der Heyden, J; Travernier, J; Plaetinck, G; Burn, P

    1996-05-28

    Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor. PMID:8643634

  15. The human fatty acid-binding protein family: Evolutionary divergences and functions

    Directory of Open Access Journals (Sweden)

    Smathers Rebecca L

    2011-03-01

    Full Text Available Abstract Fatty acid-binding proteins (FABPs are members of the intracellular lipid-binding protein (iLBP family and are involved in reversibly binding intracellular hydrophobic ligands and trafficking them throughout cellular compartments, including the peroxisomes, mitochondria, endoplasmic reticulum and nucleus. FABPs are small, structurally conserved cytosolic proteins consisting of a water-filled, interior-binding pocket surrounded by ten anti-parallel beta sheets, forming a beta barrel. At the superior surface, two alpha-helices cap the pocket and are thought to regulate binding. FABPs have broad specificity, including the ability to bind long-chain (C16-C20 fatty acids, eicosanoids, bile salts and peroxisome proliferators. FABPs demonstrate strong evolutionary conservation and are present in a spectrum of species including Drosophila melanogaster, Caenorhabditis elegans, mouse and human. The human genome consists of nine putatively functional protein-coding FABP genes. The most recently identified family member, FABP12, has been less studied.

  16. RNAcontext: a new method for learning the sequence and structure binding preferences of RNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Hilal Kazan

    Full Text Available Metazoan genomes encode hundreds of RNA-binding proteins (RBPs. These proteins regulate post-transcriptional gene expression and have critical roles in numerous cellular processes including mRNA splicing, export, stability and translation. Despite their ubiquity and importance, the binding preferences for most RBPs are not well characterized. In vitro and in vivo studies, using affinity selection-based approaches, have successfully identified RNA sequence associated with specific RBPs; however, it is difficult to infer RBP sequence and structural preferences without specifically designed motif finding methods. In this study, we introduce a new motif-finding method, RNAcontext, designed to elucidate RBP-specific sequence and structural preferences with greater accuracy than existing approaches. We evaluated RNAcontext on recently published in vitro and in vivo RNA affinity selected data and demonstrate that RNAcontext identifies known binding preferences for several control proteins including HuR, PTB, and Vts1p and predicts new RNA structure preferences for SF2/ASF, RBM4, FUSIP1 and SLM2. The predicted preferences for SF2/ASF are consistent with its recently reported in vivo binding sites. RNAcontext is an accurate and efficient motif finding method ideally suited for using large-scale RNA-binding affinity datasets to determine the relative binding preferences of RBPs for a wide range of RNA sequences and structures.

  17. Ligand-Binding Properties of the Carboxyl-Terminal Repeat Domain of Streptococcus mutans Glucan-Binding Protein A

    OpenAIRE

    Haas, Wolfgang; Banas, Jeffrey A.

    2000-01-01

    Streptococcus mutans glucan-binding protein A (GbpA) has sequence similarity in its carboxyl-terminal domain with glucosyltransferases (GTFs), the enzymes responsible for catalyzing the synthesis of the glucans to which GbpA and GTFs can bind and which promote S. mutans attachment to and accumulation on the tooth surface. It was predicted that this C-terminal region, comprised of what have been termed YG repeats, represents the GbpA glucan-binding domain (GBD). In an effort to test this hypot...

  18. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara; Briner, David; Linders, Bruce; McDonald, Joseph; Holmskov, Uffe; Head, James; Hartshorn, Kevan

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each...

  19. FK506-binding protein from adult Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae)

    Science.gov (United States)

    We successfully identified a new member of the FK-binding proteins from the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). FK-binding proteins (FKBP) function in many critical pathways needed for psyllid survival. The full length mRNA transcript provides us a new genetic target for ...

  20. Acyl-CoA binding proteins; structural and functional conservation over 2000 MYA

    DEFF Research Database (Denmark)

    Faergeman, Nils J; Wadum, Majken; Feddersen, Søren;

    2007-01-01

    -CoA binding protein, ACBP, has been proposed to play a pivotal role in the intracellular trafficking and utilization of long-chain fatty acyl-CoA esters. Depletion of acyl-CoA binding protein in yeast results in aberrant organelle morphology incl. fragmented vacuoles, multi-layered plasma membranes and...

  1. Steady-State Fluorescence Anisotropy to Investigate Flavonoids Binding to Proteins

    Science.gov (United States)

    Ingersoll, Christine M.; Strollo, Christen M.

    2007-01-01

    The steady-state fluorescence anisotropy is employed to study the binding of protein of a model protein, human serum albumin, to a commonly used flavonoid, quercetin. The experiment describes the thermodynamics, as well as the biochemical interactions of such binding effectively.

  2. A plant DNA-binding protein that recognizes 5-methylcytosine residues.

    OpenAIRE

    Zhang, D. L.; Ehrlich, K C; Supakar, P C; Ehrlich, M

    1989-01-01

    A novel, 5-methylcytosine-specific, DNA-binding protein, DBP-m, has been identified in nuclear extracts of peas. DBP-m specifically recognizes 5-methylcytosine residues in DNA without appreciable DNA sequence specificity, unlike a mammalian DNA-binding protein (MDBP), which recognizes 5-methylcytosine residues but only in a related family of 14-base-pair sequences.

  3. Identification of procollagen promoter DNA-binding proteins: effects of dexamethasone

    International Nuclear Information System (INIS)

    Glucocorticoids selectively decrease procollagen synthesis by decreasing procollagen mRNA transcription. Dexamethasone coordinately decreased total cellular type I and type III procollagen mRNAs in mouse embryonic skin fibroblasts. Since sequence specific DNA-binding proteins are known to modulate eukaryotic gene expression the authors identified in mouse fibroblasts nuclear proteins which bind to types I and III procollagen promoter DNAs. Nuclear proteins were electrophoresed, blotted onto nitrocellulose and probed with 32P-end-labeled type I and type III procollagen promoter DNAs in the presence of equimolar amounts of 32P-end-labeled vector DNA. Differences in total DNA binding were noted by the densitometric scans of the nuclear proteins. Dexamethasone treatment enhanced total DNA binding. Increasing the NaCl concentration decreased the number of promoter DNA-binding proteins without altering the relative specificity for the promoter DNAs. Promoter DNA binding to nuclear proteins was also inhibited by increasing concentrations of E. coli DNA. The number of DNA-binding proteins was greater for type III procollagen promoter DNA. The effect of dexamethasone treatment on promoter DNA binding to nuclear proteins was determined

  4. Cytosolic fatty acid-binding proteins: subjects and tools in metabolic research

    International Nuclear Information System (INIS)

    Fatty acid-binding proteins (FABPs) are major targets for specific binding of fatty acids in vivo. They constitute a widely expressed family of genetically related, small cytosolic proteins which very likely mediate intracellular transport of free long chain fatty acids. Genetic inhibition of FABP expression in vivo should therefore provide a useful tool to investigate and engineer fatty acid metabolism. (orig.)

  5. CLONING AND CHARACTERIZATION OF A NUCLEAR, SITE-SPECIFIC SSDNA BINDING-PROTEIN

    NARCIS (Netherlands)

    SMIDT, MP; RUSSCHEN, B; SNIPPE, L; WIJNHOLDS, J; AB, G

    1995-01-01

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of

  6. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    Science.gov (United States)

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  7. Viral Proteins That Bind Double-Stranded RNA: Countermeasures Against Host Antiviral Responses

    OpenAIRE

    Krug, Robert M.

    2014-01-01

    Several animal viruses encode proteins that bind double-stranded RNA (dsRNA) to counteract host dsRNA-dependent antiviral responses. This article discusses the structure and function of the dsRNA-binding proteins of influenza A virus and Ebola viruses (EBOVs).

  8. Structural Basis for a Ribofuranosyl Binding Protein: Insights into the Furanose Specific Transport

    Energy Technology Data Exchange (ETDEWEB)

    Bagaria, A.; Swaminathan, S.; Kumaran, D.; Burley, S. K.

    2011-04-01

    The ATP-binding cassette transporters (ABC-transporters) are members of one of the largest protein superfamilies, with representatives in all extant phyla. These integral membrane proteins utilize the energy of ATP hydrolysis to carry out certain biological processes, including translocation of various substrates across membranes and non-transport related processes such as translation of RNA and DNA repair. Typically, such transport systems in bacteria consist of an ATP binding component, a transmembrane permease, and a periplasmic receptor or binding protein. Soluble proteins found in the periplasm of gram-negative bacteria serve as the primary receptors for transport of many compounds, such as sugars, small peptides, and some ions. Ligand binding activates these periplasmic components, permitting recognition by the membrane spanning domain, which supports for transport and, in some cases, chemotaxis. Transport and chemotaxis processes appear to be independent of one another, and a few mutants of bifunctional periplasmic components reveal the absence of one or the other function. Previously published high-resolution X-ray structures of various periplasmic ligand binding proteins include Arabinose binding protein (ABP), Allose binding protein (ALBP), Glucose-galactose binding protein (GBP) and Ribose binding protein (RBP). Each of these proteins consists of two structurally similar domains connected by a three-stranded hinge region, with ligand buried between the domains. Upon ligand binding and release, various conformational changes have been observed. For RBP, open (apo) and closed (ligand bound) conformations have been reported and so for MBP. The closed/active form of the protein interacts with the integral membrane component of the system in both transport and chemotaxis. Herein, we report 1.9{angstrom} resolution X-ray structure of the R{sub f}BP periplasmic component of an ABC-type sugar transport system from Hahella chejuensis (UniProt Id Q2S7D2) bound to

  9. Structural Basis for a Ribofuranosyl Binding Protein: Insights into the Furanose Specific Transport

    Energy Technology Data Exchange (ETDEWEB)

    A Bagaria; D Kumaran; S Burley; S Swaminathan

    2011-12-31

    The APT-binding cassette transporters (ABC-transporters) are members of one of the largest protein superfamilies, with representatives in all extant phyla. These integral membrane proteins utilize the energy of ATP hydrolysis to carry out certain biological processes, including translocation of various substrates across membranes and nontransport related processes such as translation of RNA and DNA repair. typically, such transport systems in bacteria consist of an ATP binding component, a transmembrane permease, and a periplasmic receptor or binding protein. Soluble proteins found in the periplasm of gram-negative bacteria serve as the primary receptors for transport of many compounds, such as sugars, small peptides, and some ions. Ligand binding activates these periplasmic components, permitting recognition by the membrane spanning domain, which supports for transport, and, in some cases, chemotaxis. Transport and chemotaxis processes appear to be independent of one another, and a few mutants of bifunctional periplasmic components reveal the absence of one or the other function. Previously published high-resolution X-ray structures of various periplasmic ligand binding proteins include Arabinose binding protein (ABP), Allose binding protein (ALBP), Glucose-galactose binding protein (GBP), and Ribose binding protein (RBP). Each of these proteins consits of two structurally similar domains connected by a three-stranded hinge region, with ligand buried between the domains. Upon ligand binding and release, various conformational changes have been observed. For RBP, open (apo) and closed (ligand bound) conformations hafve been reported and so for MBP. The closed/active form of the protein interacts with the ingral membrane component of the system in both transport and chemotaxis. Herein, they report 1.9 {angstrom} resolution X-ray structure of the R{sub f}BP periplasmic component of an ABC-type sugar transport system from Hahella chejuensis (UniProt Id Q2S7D2) bound

  10. The expression of selenium-binding protein 1 is decreased in uterine leiomyoma

    Directory of Open Access Journals (Sweden)

    Quddus M Ruhul

    2010-12-01

    Full Text Available Abstract Background Selenium has been shown to inhibit cancer development and growth through the mediation of selenium-binding proteins. Decreased expression of selenium-binding protein 1 has been reported in cancers of the prostate, stomach, colon, and lungs. No information, however, is available concerning the roles of selenium-binding protein 1 in uterine leiomyoma. Methods Using Western Blot analysis and immunohistochemistry, we examined the expression of selenium-binding protein 1 in uterine leiomyoma and normal myometrium in 20 patients who had undergone hysterectomy for uterine leiomyoma. Results and Discussion The patient age ranged from 34 to 58 years with a mean of 44.3 years. Proliferative endometrium was seen in 8 patients, secretory endometrium in 7 patients, and atrophic endometrium in 5 patients. Two patients showed solitary leiomyoma, and eighteen patients revealed 2 to 5 tumors. Tumor size ranged from 1 to 15.5 cm with a mean of 4.3 cm. Both Western Blot analysis and immunohistochemistry showed a significant lower level of selenium-binding protein 1 in leiomyoma than in normal myometrium. Larger tumors had a tendency to show a lower level of selenium-binding protein 1 than smaller ones, but the difference did not reach a statistical significance. The expression of selenium-binding protein 1 was the same among patients with proliferative, secretory, and atrophic endometrium in either leiomyoma or normal myometrium. Also, we did not find a difference of selenium-binding protein 1 level between patients younger than 45 years and older patients in either leiomyoma or normal myometrium. Conclusions Decreased expression of selenium-binding protein 1 in uterine leiomyoma may indicate a role of the protein in tumorigenesis. Our findings may provide a basis for future studies concerning the molecular mechanisms of selenium-binding protein 1 in tumorigenesis as well as the possible use of selenium in prevention and treatment of uterine

  11. Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks

    Energy Technology Data Exchange (ETDEWEB)

    Mans, Ben J.; Ribeiro, Jose M.C.; Andersen, John F. (NIH)

    2008-08-19

    Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to the lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.

  12. Berberine Regulated Gck, G6pc, Pck1 and Srebp-1c Expression and Activated AMP-activated Protein Kinase in Primary Rat Hepatocytes

    Directory of Open Access Journals (Sweden)

    Yuebin Ge, Yan Zhang, Rui Li, Wei Chen, Yang Li, Guoxun Chen

    2011-01-01

    Full Text Available The effects of hormonal and dietary stimuli on hepatic glucose and lipid homeostasis include regulation of gene expression. Berberine, an effective compound in certain Chinese medicinal herbs, has been reported to lower plasma glucose and lipid levels in diabetic and hypercholesterolemic patients. We hypothesized that it may affect the expression of hepatic genes involved in glucose and lipid metabolism. The effects of berberine hydrochloride on viability, gene expression, and activation of AMP activated protein kinase (AMPK in primary hepatocytes from Sprague-Dawley (SD, Zucker lean (ZL or fatty (ZF rats were examined with MTT assay, real-time PCR, and western blotting, respectively. Berberine hydochloride at 50 µM or higher caused cytotoxic effects on hepatocytes. In SD and ZL hepatocytes, it induced Gck and suppressed G6pc expression at 10 and 25 µM, but not as potent as 1 nM insulin. Its effects on Pck1, and insulin-regulated Gck and G6pc expression depended on the hepatocyte sources and the dosage used. In ZF hepatocytes, it increased Gck, and suppressed Pck1 and G6pc expression without insulin. Its effects on Gck and G6pc, but not Pck1 expression, were additive with insulin. Berberine hydrochloride at 25 µM attenuated insulin-suppressed Pck1 (ZL/ZF cells, and insulin-induced Srebp-1c expression (SD/ZL/ZF cells, suggesting modulation of insulin action. Berberine hydrochloride did not alter these genes' mRNA stability. Its treatment caused a dose-dependent increase of phosphorylation of AMPKα, and its substrate, acetyl-CoA carboxylase, in primary hepatocytes. We conclude that berberine hydrochloride regulated the transcription of hepatic genes involved in glucose and fatty acid metabolism.

  13. Arabidopsis Pumilio protein APUM5 suppresses Cucumber mosaic virus infection via direct binding of viral RNAs

    OpenAIRE

    Huh, Sung Un; Kim, Min Jung; Paek, Kyung-Hee

    2012-01-01

    Posttranscriptional/translational regulation of gene expression is mediated by diverse RNA binding proteins and plays an important role in development and defense processes. Among the RNA-binding proteins, the mammalian Pumilio RNA-binding family (Puf) acts as posttranscriptional and translational repressors. An Arabidopsis Puf mutant, apum5-D, was isolated during a T-DNA insertional mutant screen for mutants with reduced susceptibility to Cucumber mosaic virus (CMV) infection. Interestingly,...

  14. D-fructose-binding proteins in bull seminal plasma: Isolation and characterization

    Czech Academy of Sciences Publication Activity Database

    Liberda, J.; Kraus, Marek; Ryšlavá, H.; Vlasáková, M.; Jonáková, Věra; Tichá, M.

    2001-01-01

    Roč. 47, č. 4 (2001), s. 113-119. ISSN 0015-5500 R&D Projects: GA ČR GA303/99/0357; GA ČR GV524/96/K162 Institutional research plan: CEZ:AV0Z5052915 Keywords : bull seminal plasma * non-heparin-binding and heparin-binding proteins * D- fructose -binding proteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.519, year: 2001

  15. Local conformational fluctuations can modulate the coupling between proton binding and global structural transitions in proteins

    OpenAIRE

    Whitten, Steven T; García-Moreno E., Bertrand; Hilser, Vincent J.

    2005-01-01

    Local conformational fluctuations in proteins can affect the coupling between ligand binding and global structural transitions. This finding was established by monitoring quantitatively how the population distribution in the ensemble of microstates of staphylococcal nuclease was affected by proton binding. Analysis of acid unfolding and proton-binding data with an ensemble-based model suggests that local fluctuations: (i) can be effective modulators of ligand-binding affinities, (ii) are impo...

  16. A Novel Approach for Identifying the Heme—Binding Proteins from Mouse Tissues

    Institute of Scientific and Technical Information of China (English)

    XiaoleiLi; XiaoshanWang; KangZhao; ZhengfengZhou; CaifengZhao; RenYan; LiangLin; TingtingLei; JianningYin; RongWang; ZhongshengSun; ZuyuanXu; JingyueBao; XiugingZhang; XiaoliFeng; SiqiLiu

    2003-01-01

    Heme is a key cofactor in aerobic life,both in eukaryotes and prokaryotes.Because of the high reactivity of ferrous protoporphyrin IX,the reactions of heme in cells are often carried out through heme-protein complexes.Traditionally studies of hemebinding proteins have been approached on a case by case basis,thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues.The procedure described here is aimed at profiling heme-binding proteins in mouse tissues sequentially by 1)purification of heme-binding proteins by hemeagarose,an affinity chromatographic resin;2)isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis;3)identification of heme-binding proteins by mass spectrometry.In five mouse tissues,over 600 protein spots were visualized on 2DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF,in which most proteins belong to heme related.This methodology makes it possible to globally characterize the heme-binding proteins in a biological system.

  17. RNA binding properties of the US11 protein from four primate simplexviruses

    Directory of Open Access Journals (Sweden)

    Tohme Sarah

    2011-11-01

    Full Text Available Abstract Background The protein encoded by the Us11 gene of herpes simplex viruses is a dsRNA binding protein which inhibits protein kinase R activity, thereby preventing the interferon-induced shut down of protein synthesis following viral infection. Us11 protein is not essential for infectivity in vitro and in mice in herpes simplex virus type 1 (HSV1, however this virus has a second, and apparently more important, inhibitor of PKR activity, the γ134.5 protein. Recently sequenced simian simplexviruses SA8, HVP2 and B virus do not have an ORF corresponding to the γ134.5 protein, yet they have similar, or greater, infectivity as HSV1 and HSV2. Methods We have expressed the US11 proteins of the simplexviruses HSV1, HSV2, HVP2 and B virus and measured their abilities to bind dsRNA, in order to investigate possible differences that could complement the absence of the γ134.5 protein. We employed a filter binding technique that allows binding of the Us11 protein under condition of excess dsRNA substrate and therefore a measurement of the true Kd value of Us11-dsRNA binding. Results and Conclusions The results show a Kd of binding in the range of 0.89 nM to 1.82 nM, with no significant difference among the four Us11 proteins.

  18. A Novel Approach for Identifying the Heme-Binding Proteins from Mouse Tissues

    Institute of Scientific and Technical Information of China (English)

    Xiaolei Li; Rong Wang; Zhongsheng Sun; Zuyuan Xu; Jingyue Bao; Xiuqing Zhang; Xiaoli Feng; Siqi Liu; Xiaoshan Wang; Kang Zhao; Zhengfeng Zhou; Caifeng Zhao; Ren Yan; Liang Lin; Tingting Lei; Jianning Yin

    2003-01-01

    Heme is a key cofactor in aerobic life, both in eukaryotes and prokaryotes. Because of the high reactivity of ferrous protoporphyrin IX, the reactions of heme in cells are often carried out through heme-protein complexes. Traditionally studies of hemebinding proteins have been approached on a case by case basis, thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues. The procedure described here is aimed at profiling hemne-binding proteins in mouse tissues sequentially by 1) purification of heme-binding proteins by hemeagarose, an affinity chromatographic resin; 2) isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis; 3) identification of heme-binding proteins by mass spectrometry. In five mouse tissues, over 600 protein spots were visualized on 2DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF, in which most proteins belong to heme related. This methodology makes it possible to globally characterize the heme-binding proteins in a biological system.

  19. A recombinant triblock protein polymer with dispersant and binding properties for digital printing.

    Science.gov (United States)

    Qi, Min; O'Brien, John P; Yang, Jianjun

    2008-01-01

    A structured triblock protein was designed to explore the potential of engineered peptides to function as high-performance ink dispersants and binders. The protein consists of three functional elements, including a pigment binding domain, a hydrophilic linker, and a printing surface binding domain. To construct such a chimeric protein, a carbon black binding peptide, FHENWPS, and a cellulose binding peptide, THKTSTQRLLAA, were identified from phage display libraries through biopanning, based on their strong and specific binding affinities to carbon black and cellulose. They were used as carbon black and cellulose binding domains, respectively, in a recombinant triblock protein. A linker sequence, PTPTPTPTPTPTPTPTPTPTPTP, was adapted from endoglucanase A of the bacterium Cellulomonas fimi, as a small, rigid, and hydrophilic interdomain linker. When incorporated into the triblock structure between the carbon black and cellulose binding sequences, the linker sufficiently isolates these two elements and allows dual binding activity. The structured triblock protein was shown to disperse carbon black particles and attach it to paper surfaces. Thus, the utility of structured proteins having useful dispersant and binding properties for digital printing inks was demonstrated. PMID:17972282

  20. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

  1. A rapid and simple assay for growth hormone-binding protein activity in human plasma

    International Nuclear Information System (INIS)

    The newly discovered circulating growth hormone binding proteins dictate a re-evaluation of the state of GH in plasma in health and disease as the binding proteins are known to affect GH metabolism and action. We describe a rapid and simple GH-binding assay that allows determination of free and complexed plasma GH, as well as GH-binding protein activity as an index of GH-binding protein levels, with relative ease. The method is based on incubation of plasma with 125I-GH and separation of bound from free GH on small DEAE-cellulose columns; it can be used on a large scale for routine determinations. The results obtained by this method are comparable to those obtained with the previously used slow and more cumbersome gel filtration technique. Initial data obtained in normal subject and certain disease states show that the bound fraction of plasma GH is similar in men, women and children, is unaffected by pregnancy or acute infection, but is marginally decreased in liver cirrhosis. In acromegaly, binding protein activity also appears normal when allowance is made for partial saturation of the binding proteins by the high prevailing GH levels. The technique we describe should facilitate investigations of normal and abnormal regulation of the GH binding proteins. (author)

  2. Serotonin binding in vitro by releasable proteins from human blood platelets

    International Nuclear Information System (INIS)

    Among the substances released from human blood platelets are serotonin and various proteins. It was hypothesized that one of these proteins binds serotonin and that serotonin might be important to the protein's function or that the protein might be important to serotonin's function. Two platelet-specific proteins, platelet factor 4 (PF4) and β-thromboglobulin (βTG) were found to bind serotonin in vitro. Endogenous PF4 was isolated by serotonin-affinity chromatography and was identified by radioimmunoassay. Purified [125I] -PF4 and native PF4 bound to and eluted from a serotonin-affinity column similarly. Ultrafiltration of the homologous protein, βTG, with [14C]-serotonin demonstrated binding of about 8 moles serotonin per mole tetrameric βTG with a dissociation constant of about 4 X 10(sup-8) M. Equilibrium dialysis of PF4 with radiolabelled serotonin was attempted, but no binding constant values were obtained because serotonin apparently bound to the dialysis membrane. Since EDTA was one of the two agents that eluted PF4 from the serotonin-affinity gel, calcium binding by PF4 was investigated by equilibrium dialysis. Evidence was obtained for positively cooperative binding of calcium ions by PF4. It is concluded that PF4 and βTG bind serotonin in vitro, that they may also bind in vivo when platelets undergo release, and that the functions of serotonin, PF4 and βTG may be mediated in part by serotonin-protein associations

  3. Enhancement of rabbit protein S anticoagulant cofactor activity in vivo by modulation of the protein S C4B binding protein interaction.

    OpenAIRE

    Weinstein, R E; Walker, F. J.

    1990-01-01

    The carboxy-terminal region of protein S has been recently been observed to be involved in the interaction between protein S and C4b-binding protein (Walker, F. J. 1989. J. Biol. Chem. 264:17645-17658). A synthetic peptide, GVQLDLDEAI, corresponding to that region of protein S has been used to investigate the protein S/C4b-binding protein interaction in vitro and in vivo. Rabbit activated protein C possesses species-specific anticoagulant activity for which rabbit protein S functions as a cof...

  4. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    Science.gov (United States)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  5. Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins.

    Science.gov (United States)

    Meek, Sarah E M; Lane, William S; Piwnica-Worms, Helen

    2004-07-30

    14-3-3 proteins regulate the cell division cycle and play a pivotal role in blocking cell cycle advancement after activation of the DNA replication and DNA damage checkpoints. Here we describe a global proteomics analysis to identify proteins that bind to 14-3-3s during interphase and mitosis. 14-3-3-binding proteins were purified from extracts of interphase and mitotic HeLa cells using specific peptide elution from 14-3-3 zeta affinity columns. Proteins that specifically bound and eluted from the affinity columns were identified by microcapillary high pressure liquid chromatography tandem mass spectrometry analysis. Several known and novel 14-3-3-interacting proteins were identified in this screen. Identified proteins are involved in cell cycle regulation, signaling, metabolism, protein synthesis, nucleic acid binding, chromatin structure, protein folding, proteolysis, nucleolar function, and nuclear transport as well as several other cellular processes. In some cases 14-3-3 binding was cell cycle-dependent, whereas in other cases the binding was shown to be cell cycle-independent. This study adds to the growing list of human 14-3-3-binding proteins and implicates a role for 14-3-3 proteins in a plethora of essential biological processes. PMID:15161933

  6. Putative hAPN receptor binding sites in SARS_CoV spike protein

    Institute of Scientific and Technical Information of China (English)

    YUXiao-Jing; LUOCheng; LinJian-Cheng; HAOPei; HEYou-Yu; GUOZong-Ming; QINLei; SUJiong; LIUBo-Shu; HUANGYin; NANPeng; LIChuan-Song; XIONGBin; LUOXiao-Min; ZHAOGuo-Ping; PEIGang; CHENKai-Xian; SHENXu; SHENJian-Hua; ZOUJian-Ping; HEWei-Zhong; SHITie-Liu; ZHONGYang; JIANGHua-Liang; LIYi-Xue

    2003-01-01

    AIM:To obtain the information of ligand-receptor binding between thd S protein of SARS_CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS: On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites sites in the S protein of SARS_CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS_CoV in validating the bioinformatics predictions. RESULTS:Possible binding sites in the SARS_CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS_CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION:CD13 may be a possible receptor of the SARS_CoV S protein which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome.

  7. Mutations in the RNA Binding Domain of Stem-Loop Binding Protein Define Separable Requirements for RNA Binding and for Histone Pre-mRNA Processing

    OpenAIRE

    Dominski, Zbigniew; Erkmann, Judith A.; Greenland, John A.; Marzluff, William F

    2001-01-01

    Expression of replication-dependent histone genes at the posttranscriptional level is controlled by stem-loop binding protein (SLBP). One function of SLBP is to bind the stem-loop structure in the 3′ untranslated region of histone pre-mRNAs and facilitate 3′ end processing. Interaction of SLBP with the stem-loop is mediated by the centrally located RNA binding domain (RBD). Here we identify several highly conserved amino acids in the RBD mutation of which results in complete or substantial lo...

  8. Phenanthrene binding by humic acid–protein complexes as studied by passive dosing technique

    International Nuclear Information System (INIS)

    This work investigated the binding behavior of phenanthrene by humic acids (HA-2 and HA-5), proteins (bovine serum albumin (BSA)), lysozyme and pepsin), and their complexes using a passive dosing technique. All sorption isotherms were fitted well with Freundlich model and the binding capability followed an order of HA-5 > HA-2 > BSA > pepsin > lysozyme. In NaCl solution, phenanthrene binding to HA-BSA complexes was much higher than the sum of binding to individual HA and BSA, while there was no enhancement for HA-pepsin. Positively charged lysozyme slightly lowered phenanthrene binding on both HAs due to strong aggregation of HA-lysozyme complexes, leading to reduction in the number of binding sites. The binding enhancement by HA-BSA was observed under all tested ion species and ionic strengths. This enhancement can be explained by unfolding of protein, reduction of aggregate size and formation of HA-BSA complexes with favorable conformations for binding phenanthrene. Highlights: • Phenanthrene binding capability followed an order: HA-5>HA-2>BSA>pepsin>lysozyme. • Phenanthrene binding to HA-BSA was enhanced relative to individual HA and BSA. • Binding enhancement to HA-BSA was observed under all tested solution conditions. • The enhancement is related to BSA unfolding, size reduction and HA-BSA complexation. -- Phenanthrene binding to HA-BSA complexes is much higher than the sum to individual HA and BSA while there was no binding enhancement to HA-pepsin or HA-lysozyme

  9. Insulin-like growth factors, insulin-like growth factor-binding proteins, insulin-like growth factor-binding protein-3 protease, and growth hormone-binding protein in lipodystrophic human immunodeficiency virus-infected patients

    DEFF Research Database (Denmark)

    Haugaard, Steen B; Andersen, Ove; Hansen, Birgitte Rønde;

    2004-01-01

    Human immunodeficiency virus (HIV)-lipodystrophy is associated with impaired growth hormone (GH) secretion. It remains to be elucidated whether insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), IGFBP-3 protease, and GH-binding protein (GHBP) are abnormal in HIV......-lipodystrophy. These parameters were measured in overnight fasting serum samples from 16 Caucasian males with HIV-lipodystrophy (LIPO) and 15 Caucasian HIV-infected males without lipodystrophy (NONLIPO) matched for age, weight, duration of HIV infection, and antiretroviral therapy. In LIPO, abdominal fat mass and insulin...

  10. Ephemeral protein binding to DNA shapes stable nuclear bodies and chromatin domains

    CERN Document Server

    Brackley, C A; Michieletto, D; Mouvet, F; Cook, P R; Marenduzzo, D

    2016-01-01

    Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear "bodies" exchange rapidly with the soluble pool whilst the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins; these proteins switch between two states -- active (binding) and inactive (non-binding). This system provides a model for any DNA-binding protein that can be modified post-translationally to change its affinity for DNA (e.g., like the phosphorylation of a transcription factor). Due to this out-of-equilibrium process, proteins spontaneously assemble into clusters of self-limiting size, as individual proteins in a cluster exchange with the soluble pool with kinetics like those seen in photo-bleaching experiments. This behavior contrasts sharply with that exhibited by "equilibrium", or non-switching, proteins that exis...

  11. Identification of CP12 as a Novel Calcium-Binding Protein in Chloroplasts

    Directory of Open Access Journals (Sweden)

    Agostinho Gomes Rocha

    2013-08-01

    Full Text Available Calcium plays an important role in the regulation of several chloroplast processes. However, very little is still understood about the calcium fluxes or calcium-binding proteins present in plastids. Indeed, classical EF-hand containing calcium-binding proteins appears to be mostly absent from plastids. In the present study we analyzed the stroma fraction of Arabidopsis chloroplasts for the presence of novel calcium-binding proteins using 2D-PAGE separation followed by calcium overlay assay. A small acidic protein was identified by mass spectrometry analyses as the chloroplast protein CP12 and the ability of CP12 to bind calcium was confirmed with recombinant proteins. CP12 plays an important role in the regulation of the Calvin-Benson-Bassham Cycle participating in the assembly of a supramolecular complex between phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase, indicating that calcium signaling could play a role in regulating carbon fixation.

  12. In vitro protein binding of liraglutide in human plasma determined by reiterated stepwise equilibrium dialysis

    Science.gov (United States)

    Plum, Anne; Jensen, Lisbeth Bjerring; Kristensen, Jesper Bøggild

    2013-01-01

    Liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue approved for the treatment of type 2 diabetes. It is based on human GLP-1 with the addition of a 16-carbon fatty acid, which facilitates binding to plasma proteins, thus prolonging the elimination half-life and allowing once-daily administration. It has not been possible to quantify liraglutide protein binding by ultrafiltration (the usual method of choice), as the lipophilic molecule becomes trapped in the filter membrane. Therefore, the aim of this study was to develop a methodology that could determine the extent of liraglutide binding to plasma proteins in vitro. We report here the details of a novel reiterated stepwise equilibrium dialysis assay that has successfully been used to quantify liraglutide plasma protein binding. The assay allowed quantification of liraglutide binding to proteins in purified plasma protein solutions and human plasma samples and was effective at plasma dilutions as low as 5%. At a clinically relevant liraglutide concentration (104 pM), greater than 98.9% of liraglutide was bound to protein. Specific binding to human serum albumin and α1-acid glycoprotein was 99.4% and 99.3%, respectively. The novel methodology described herein could have an application in the quantification of plasma protein binding of other highly lipophilic drug molecules. PMID:23853127

  13. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    Science.gov (United States)

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J.; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2012-10-01

    Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

  14. Hemoglobin binding activity and hemoglobin-binding protein of prevotella nigrescens

    OpenAIRE

    Miyashita M; Oishi S; Kiso A; Kikuchi Y; Ueda O; Hirai K; Shibata Y; Fujimura S

    2010-01-01

    Abstract Prevotella nigrescens, lacking siderophores was found to bind to the hemoproteins. The binding was observed also in the envelope which was prepared by sonication of the cell. The binding occurred in the pH-dependent manner; the binding was observed below neutral pHs of the incubation mixtures but only slightly observed in the neutral and alkaline pHs. Furthermore, hemoglobin bound to the envelope was dissociated at high pHs buffers. Maximum amounts of hemoglobin bound to 1 mg envelop...

  15. Elucidation of binding mechanism and identification of binding site for an anti HIV drug, stavudine on human blood proteins.

    Science.gov (United States)

    Sandhya, B; Hegde, Ashwini H; Seetharamappa, J

    2013-05-01

    The binding of stavudine (STV) to two human blood proteins [human hemoglobin (HHb) and human serum albumin (HSA)] was studied in vitro under simulated physiological conditions by spectroscopic methods viz., fluorescence, UV absorption, resonance light scattering, synchronous fluorescence, circular dichroism (CD) and three-dimensional fluorescence. The binding parameters of STV-blood protein were determined from fluorescence quenching studies. Stern-Volmer plots indicated the presence of static quenching mechanism in the interaction of STV with blood proteins. The values of n close to unity indicated that one molecule of STV bound to one molecule of blood protein. The binding process was found to be spontaneous. Analysis of thermodynamic parameters revealed the presence of hydrogen bond and van der Waals forces between protein and STV. Displacement experiments indicated the binding of STV to Sudlow's site I on HSA. Secondary structures of blood proteins have undergone changes upon interaction with STV as evident from the reduction of α-helices (from 46.11% in free HHb to 38.34% in STV-HHb, and from 66.44% in free HSA to 52.26% in STV-HSA). Further, the alterations in secondary structures of proteins in the presence of STV were confirmed by synchronous and 3D-fluorescence spectral data. The distance between the blood protein (donor) and acceptor (STV) was found to be 5.211 and 5.402 nm for STV-HHb and STV-HSA, respectively based on Föster's non-radiative energy transfer theory. Effect of some metal ions was also investigated. The fraction of STV bound to HSA was found to be 87.8%. PMID:23275205

  16. Expression of liver fatty acid binding protein in hepatocellular carcinoma.

    Science.gov (United States)

    Cho, Soo-Jin; Ferrell, Linda D; Gill, Ryan M

    2016-04-01

    Loss of expression of liver fatty acid binding protein (LFABP) by immunohistochemistry has been shown to be characteristic of a subset of hepatocellular adenomas (HCAs) in which HNF1A is inactivated. Transformation to hepatocellular carcinoma is thought to be a very rare phenomenon in the HNF1A-inactivated variant of HCA. However, we recently observed 2 cases at our institution, 1 definite hepatocellular carcinoma and 1 possible hepatocellular carcinoma, with loss of LFABP staining, raising the possibility that LFABP down-regulation may be associated with hepatocellular carcinogenesis. Our aim was to evaluate hepatocellular carcinomas arising in various backgrounds and with varying degrees of differentiation for loss of LFABP staining. Twenty total cases of hepatocellular carcinoma were examined. Thirteen cases arose in a background of cirrhosis due to hepatitis C (n = 8) or steatohepatitis (n = 5); 7 cases arose in a noncirrhotic background, with 2 cases arising within HNF1A-inactivated variant HCA and 2 cases arising within inflammatory variant HCA. Complete loss of expression of LFABP was seen in 6 of 20 cases, including 2 cases of hepatocellular carcinoma arising within HNF1A-inactivated variant HCA. Thus, loss of staining for LFABP appears to be common in hepatocellular carcinoma and may be seen in well-differentiated hepatocellular carcinoma. Therefore, LFABP loss should not be interpreted as evidence for hepatocellular adenoma over carcinoma, when other features support a diagnosis of hepatocellular carcinoma. The findings raise consideration for a role of HNF1A inactivation in hepatocellular carcinogenesis, particularly in less differentiated tumors. PMID:26997447

  17. Proteomic Screening Method for Phosphopeptide Motif Binding Proteins Using Peptide Libraries

    OpenAIRE

    Christofk, Heather R.; Wu, Ning; Cantley, Lewis C.; John M. Asara

    2011-01-01

    Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially-degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC/MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pTh...

  18. Identification and characterization of amylase binding protein C (AbpC) from Streptococcus mitis NS51

    OpenAIRE

    Vorrasi, John; Chaudhuri, Biswendu; Haase, Elaine M.; Scannapieco, Frank A.

    2010-01-01

    A substantial proportion of the streptococcal species found in dental plaque biofilms are able to interact with the abundant salivary enzyme α-amylase. These streptococci produce proteins that specifically bind amylase. An important plaque species, Streptococcus mitis, secretes a 36-kDa amylase binding protein into the extracellular milieu. Proteins precipitated from S. mitis NS51 cell culture supernatant by the addition of purified salivary amylase were separated by SDS-PAGE, transferred to ...

  19. Computational analysis of protein-ligand binding : from single continuous trajectories to multiple parallel simulations

    OpenAIRE

    Thorsteinsdottir, Holmfridur B.

    2010-01-01

    The interaction of proteins with other proteins or small molecules is essential for biological functions. Understanding the molecular basis of protein-ligand binding is of a vast interest for drug discovery, and computational methods to estimate proteinligand binding are starting to play an increasingly important role. In order to apply atomistic computational methods to the drug discovery process it is necessary to have accurate three-dimensional structures of the target prote...

  20. Multiple GTP-binding proteins participate in clathrin-coated vesicle- mediated endocytosis

    OpenAIRE

    1993-01-01

    We have examined the effects of various agonists and antagonists of GTP- binding proteins on receptor-mediated endocytosis in vitro. Stage- specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to ...

  1. Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1.

    OpenAIRE

    Moreland, R B; Montross, L; Garcea, R L

    1991-01-01

    The major capsid protein of polyomavirus, VP1, has been expression cloned in Escherichia coli, and the recombinant VP1 protein has been purified to near homogeneity (A. D. Leavitt, T. M. Roberts, and R. L. Garcea, J. Biol. Chem. 260:12803-12809, 1985). With this recombinant protein, a nitrocellulose filter transfer assay was developed for detecting DNA binding to VP1 (Southwestern assay). In optimizing conditions for this assay, dithiothreitol was found to inhibit DNA binding significantly. W...

  2. Loss of liver FA binding protein significantly alters hepatocyte plasma membrane microdomains[S

    OpenAIRE

    McIntosh, Avery L.; Atshaves, Barbara P.; Storey, Stephen M.; Landrock, Kerstin K.; Landrock, Danilo; Martin, Gregory G.; Kier, Ann B.; Schroeder, Friedhelm

    2012-01-01

    Although lipid-rich microdomains of hepatocyte plasma membranes serve as the major scaffolding regions for cholesterol transport proteins important in cholesterol disposition, little is known regarding intracellular factors regulating cholesterol distribution therein. On the basis of its ability to bind cholesterol and alter hepatic cholesterol accumulation, the cytosolic liver type FA binding protein (L-FABP) was hypothesized to be a candidate protein regulating these microdomains. Compared ...

  3. Binding of the polypyrimidine tract-binding protein-associated splicing factor (PSF) to the hepatitis delta virus RNA

    International Nuclear Information System (INIS)

    The hepatitis delta virus (HDV) has a very limited protein coding capacity and must rely on host proteins for its replication. A ribonucleoprotein complex was detected following UV cross-linking between HeLa nuclear proteins and an RNA corresponding to the right terminal stem-loop domain of HDV genomic RNA. Mass spectrometric analysis of the complex revealed the polypyrimidine tract-binding protein-associated splicing factor (PSF) as a novel HDV RNA-interacting protein. Co-immunoprecipitation demonstrated the interaction between HDV RNA and PSF both in vitro in HeLa nuclear extract and in vivo within HeLa cells containing both polarities of the HDV genome. Analysis of the binding of various HDV-derived RNAs to purified, recombinant PSF further confirmed the specificity of the interaction and revealed that PSF directly binds to the terminal stem-loop domains of both polarities of HDV RNA. Our findings provide evidence of the involvement of a host mRNA processing protein in the HDV life cycle

  4. Autoradiographic characterization of (+-)-1-(2,5-dimethoxy-4-[125I] iodophenyl)-2-aminopropane ([125I]DOI) binding to 5-HT2 and 5-HT1c receptors in rat brain

    International Nuclear Information System (INIS)

    The 5-HT2 (serotonin) receptor has traditionally been labeled with antagonist radioligands such as [3H]ketanserin and [3H]spiperone, which label both agonist high-affinity (guanyl nucleotide-sensitive) and agonist low-affinity (guanyl nucleotide-insensitive) states of this receptor. The hallucinogen 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) is an agonist which labels the high-affinity guanyl nucleotide-sensitive state of brain 5-HT2 receptors selectively. In the present study, conditions for autoradiographic visualization of (+/-)-[125I]DOI-labeled 5-HT2 receptors were optimized and binding to slide-mounted sections was characterized with respect to pharmacology, guanyl nucleotide sensitivity and anatomical distribution. In slide-mounted rat brain sections (+/-)-[125I]DOI binding was saturable, of high affinity (KD approximately 4 nM) and displayed a pharmacologic profile typical of 5-HT2 receptors. Consistent with coupling of 5-HT2 receptors in the high-affinity state to a guanyl nucleotide regulatory protein, [125I]DOI binding was inhibited by guanyl nucleotides but not by adenosine triphosphate. Patterns of autoradiographic distribution of [125I]DOI binding to 5-HT2 receptors were similar to those seen with [3H]ketanserin- and [125I]-lysergic acid diethylamide-labeled 5-HT2 receptors. However, the density of 5-HT2 receptors labeled by the agonist [125I]DOI was markedly lower (30-50%) than that labeled by the antagonist [3H]ketanserin. High densities of [125I]DOI labeling were present in olfactory bulb, anterior regions of cerebral cortex (layer IV), claustrum, caudate putamen, globus pallidus, ventral pallidum, islands of Calleja, mammillary nuclei and inferior olive. Binding in hippocampus, thalamus and hypothalamus was generally sparse

  5. Guanine nucleotide binding proteins in zucchini seedlings: Characterization and interactions with the NPA receptor

    International Nuclear Information System (INIS)

    A microsomal membrane preparation from hypocotyls of dark-grown Cucurbita pepo L. seedlings contains specific high-affinity binding sites for the non-hydrolyzable GTP analog guanosine 5'-[γ-thio] triphosphate (GTP-γ-S). Both the binding affinity and the pattern of binding specificity for GTP and GTP analogs are similar to animal G-proteins, and two zucchini membrane proteins are recognized in western blots by antiserum specific for the σ subunit of platelet Gs protein. GTP-γ-S can increase specific naphthylphthalamic acid (NPA) binding in zucchini microsomal membrane preparations, with its stimulation increasing with large tissue age. Al+3 and F- agents known to activate G-proteins - decreased NPA specific binding by ca. 15%. In tests of in vitro auxin transport employing zucchini plasma membrane vesicles, AlF-4 strongly inhibited 3H-indoleacetic acid nor accumulation; GTP-γ-S effects on this system will be discussed

  6. Bioluminescent Ligand-Receptor Binding Assays for Protein or Peptide Hormones.

    Science.gov (United States)

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    Bioluminescence has been widely used in biomedical research due to its high sensitivity, low background, and broad linear range. In recent studies, we applied bioluminescence to ligand-receptor binding assays for some protein or peptide hormones based on a newly developed small monomeric Nanoluciferase (NanoLuc) reporter that has the so far brightest bioluminescence. The conventional ligand-receptor binding assays rely on radioligands that have drawbacks, such as radioactive hazards and short shelf lives. In contrast, the novel bioluminescent binding assays use the NanoLuc-based protein or peptide tracers that are safe, stable, and ultrasensitive. Thus, the novel bioluminescent ligand-receptor binding assay would be applied to more and more protein or peptide hormones for ligand-receptor interaction studies in future. In the present article, we provided detailed protocols for setting up the novel bioluminescent ligand-receptor binding assays using two representative protein hormones as examples. PMID:27424896

  7. Annexins V and VI: major calcium-dependent atrial secretory granule-binding proteins.

    Science.gov (United States)

    Doubell, A F; Bester, A J; Thibault, G

    1991-11-01

    Atrial natriuretic peptide is stored by atrial myocytes in secretory granules, known as atrial specific granules, and is released from these granules by exocytosis. We have isolated a group of atrial proteins by affinity chromatography that bind to atrial specific granules in a calcium-dependent manner. The two major proteins isolated (32.5 kd and 67 kd) are calcium-binding proteins and have been identified as annexins V and VI by immunoblotting with specific antisera. The calcium dependence of their binding to atrial specific granules has been characterized in vitro and indicates that this interaction takes place at micromolar levels of calcium. In addition, the group of proteins isolated includes another calcium-binding protein of 20 kd, as well as GTP-binding proteins of 22 to 26 kd. Membrane interactions during exocytosis are presumably mediated by the interaction of specific proteins with the granule membrane. The properties of the proteins described here, and their ability to bind to atrial specific granules in a calcium-dependent manner, make them likely candidates in the search for regulatory proteins mediating atrial natriuretic peptide secretion. PMID:1834552

  8. Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens.

    Science.gov (United States)

    MacIntosh, S C; Stone, T B; Jokerst, R S; Fuchs, R L

    1991-10-15

    A laboratory-selected colony of Heliothis virescens displaying a 20- to 70-fold level of resistance to Bacillus thuringiensis proteins was evaluated to identify mechanism(s) of resistance. Brush-border membrane vesicles were isolated from larval midgut epithelium from the susceptible and resistant strains of H. virescens. Two B. thuringiensis proteins, CryIA(b) and CryIA(c), were iodinated and shown to specifically bind to brush-border membrane vesicles of both insect strains. Multiple changes in the receptor-binding parameters were seen in the resistant strain as compared with the susceptible strain. A 2- to 4-fold reduction in binding affinity was accompanied by a 4- to 6-fold increase in binding-site concentration for both proteins. Although these two B. thuringiensis proteins competed for the same high-affinity binding site, competition experiments revealed different receptor specificity toward these proteins in the resistant H. virescens line. The H. virescens strains were not sensitive to a coleopteran-active protein, CryIIIA, nor did these proteins compete with the CryIA proteins for binding. Complexity of the mechanism of resistance is consistent with the complex mode of action of B. thuringiensis proteins. PMID:1924353

  9. Ligand-binding properties of the carboxyl-terminal repeat domain of Streptococcus mutans glucan-binding protein A.

    Science.gov (United States)

    Haas, W; Banas, J A

    2000-02-01

    Streptococcus mutans glucan-binding protein A (GbpA) has sequence similarity in its carboxyl-terminal domain with glucosyltransferases (GTFs), the enzymes responsible for catalyzing the synthesis of the glucans to which GbpA and GTFs can bind and which promote S. mutans attachment to and accumulation on the tooth surface. It was predicted that this C-terminal region, comprised of what have been termed YG repeats, represents the GbpA glucan-binding domain (GBD). In an effort to test this hypothesis and to quantitate the ligand-binding specificities of the GbpA GBD, several fusion proteins were generated and tested by affinity electrophoresis or by precipitation of protein-ligand complexes, allowing the determination of binding constants. It was determined that the 16 YG repeats in GbpA comprise its GBD and that GbpA has a greater affinity for dextran (a water-soluble form of glucan) than for mutan (a water-insoluble form of glucan). Placement of the GBD at the carboxyl terminus was necessary for maximum glucan binding, and deletion of as few as two YG repeats from either end of the GBD reduced the affinity for dextran by over 10-fold. Interestingly, the binding constant of GbpA for dextran was 34-fold higher than that calculated for the GBDs of two S. mutans GTFs, one of which catalyzes the synthesis of water-soluble glucan and the other of which catalyzes the synthesis of water-insoluble glucan. PMID:10633107

  10. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

    Directory of Open Access Journals (Sweden)

    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

  11. Urinary excretion of fatty acid-binding protein 4 is associated with albuminuria and renal dysfunction.

    Directory of Open Access Journals (Sweden)

    Yusuke Okazaki

    Full Text Available Fatty acid-binding protein 4 (FABP4/A-FABP/aP2 is expressed in not only adipocytes and macrophages but also peritubular capillaries in the normal kidney. We recently demonstrated that ectopic expression of FABP4, but not FABP1 known as liver FABP (L-FABP, in the glomerulus is associated with progression of proteinuria and renal dysfunction. However, urinary excretion of FABP4 has not been investigated.Subjects who participated in the Tanno-Sobetsu Study, a study with a population-based cohort design, in 2011 (n = 392, male/female: 166/226 were enrolled. Urinary FABP4 (U-FABP4 and urinary albumin-to-creatinine ratio (UACR were measured. Change in estimated glomerular filtration rate (eGFR was followed up one year later.In 93 (23.7% of the 392 subjects, U-FABP4 level was below the sensitivity of the assay. Subjects with undetectable U-FABP4 were younger and had lower UACR and higher eGFR levels than subjects with measurable U-FABP4. U-FABP4 level was positively correlated with age, systolic blood pressure and levels of serum FABP4 (S-FABP4, triglycerides, hemoglobin A1c (HbA1c, urinary FABP1 (U-FABP1 and UACR (r = 0.360, p<0.001. Age, S-FABP4, U-FABP1 and UACR were independent predictors of U-FABP4. On the other hand, systolic blood pressure, HbA1c and U-FABP4 were independently correlated with UACR. Reduction in eGFR after one year was significantly larger in a group with the highest tertile of baseline U-FABP4 than a group with the lowest tertile.Urinary FABP4 level is independently correlated with level of albuminuria and possibly predicts yearly decline of eGFR. U-FABP4 would be a novel biomarker of glomerular damage.

  12. Exploring the composition of protein-ligand binding sites on a large scale.

    Directory of Open Access Journals (Sweden)

    Nickolay A Khazanov

    Full Text Available The residue composition of a ligand binding site determines the interactions available for diffusion-mediated ligand binding, and understanding general composition of these sites is of great importance if we are to gain insight into the functional diversity of the proteome. Many structure-based drug design methods utilize such heuristic information for improving prediction or characterization of ligand-binding sites in proteins of unknown function. The Binding MOAD database if one of the largest curated sets of protein-ligand complexes, and provides a source of diverse, high-quality data for establishing general trends of residue composition from currently available protein structures. We present an analysis of 3,295 non-redundant proteins with 9,114 non-redundant binding sites to identify residues over-represented in binding regions versus the rest of the protein surface. The Binding MOAD database delineates biologically-relevant "valid" ligands from "invalid" small-molecule ligands bound to the protein. Invalids are present in the crystallization medium and serve no known biological function. Contacts are found to differ between these classes of ligands, indicating that residue composition of biologically relevant binding sites is distinct not only from the rest of the protein surface, but also from surface regions capable of opportunistic binding of non-functional small molecules. To confirm these trends, we perform a rigorous analysis of the variation of residue propensity with respect to the size of the dataset and the content bias inherent in structure sets obtained from a large protein structure database. The optimal size of the dataset for establishing general trends of residue propensities, as well as strategies for assessing the significance of such trends, are suggested for future studies of binding-site composition.

  13. Role of Arabidopsis Pumilio RNA binding protein 5 in virus infection

    OpenAIRE

    Un Huh, Sung; Paek, Kyung-Hee

    2013-01-01

    Regulation of gene expression is mediated by diverse RNA binding proteins which play important roles in development and defense processes. Pumilio/FBF (Puf) protein in mammals functions as a posttranscriptional/translational repressor by binding to the 3′ UTR regions of its target mRNAs. Previous study reported that APUM5 provides protection against CMV infection by directly binding to CMV RNAs in Arabidopsis. CMV RNAs contain putative Pumilio-binding motifs and APUM5 bound to the 3′ UTR and ...

  14. Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems.

    Science.gov (United States)

    Fielding, Lee; Rutherford, Samantha; Fletcher, Dan

    2005-06-01

    The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems. PMID:15816062

  15. Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells

    Directory of Open Access Journals (Sweden)

    Tao Zhang

    2015-11-01

    Full Text Available The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS. More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1, heat shock 70 kDa protein 9 (HSPA9 and pyruvate kinase M2 (PKM2, were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

  16. Determination of low isotopic enrichment of L-[1-C-13]valine by gas chromatography combustion isotope ratio mass spectrometry : a robust method for measuring protein fractional synthetic rates in vivo

    NARCIS (Netherlands)

    Reijngoud, DJ; Hellstern, G; Elzinga, H; de Sain-van der Velden, MG; Okken, A; Stellaard, F

    1998-01-01

    A method was developed for measuring protein fractional synthetic rates using the N-methoxycarbonylmethyl ester (MCM) derivative of L-[1-C-13]valine and on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The derivatization procedure can be performed rapidly and GC sep

  17. Enterocyte Fatty Acid Binding Proteins (FABPs): Different Functions of Liver- and Intestinal- FABPs in the Intestine

    Science.gov (United States)

    Gajda, Angela M.; Storch, Judith

    2014-01-01

    SUMMARY Fatty acid binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both Liver- (LFABP; FABP1) and Intestinal-fatty acid binding proteins (IFABP; FABP2) are expressed. These proteins display high affinity binding for long chain fatty acids (FA) and other hydrophobic ligands, thus they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences in ligand binding stoichiometry and specificity, and in mechanisms of FA transfer to membranes, and it has been hypothesized that LFABP and IFABP have difference functions in the enterocyte. Studies directly comparing LFABP- and IFABP-null mice have revealed markedly different phenotypes, indicating that these proteins indeed have different functions in intestinal lipid metabolism and whole body energy homeostasis. In this review, we discuss the evolving knowledge of the functions of LFABP and IFABP in the intestinal enterocyte. PMID:25458898

  18. Docking of the Periplasmic FecB Binding Protein to the FecCD Transmembrane Proteins in the Ferric Citrate Transport System of Escherichia coli▿

    OpenAIRE

    Braun, Volkmar; Herrmann, Christina

    2007-01-01

    Citrate-mediated iron transport across the cytoplasmic membrane is catalyzed by an ABC transporter that consists of the periplasmic binding protein FecB, the transmembrane proteins FecC and FecD, and the ATPase FecE. Salt bridges between glutamate residues of the binding protein and arginine residues of the transmembrane proteins are predicted to mediate the positioning of the substrate-loaded binding protein on the transmembrane protein, based on the crystal structures of the ABC transporter...

  19. The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.; Gomes Guimaraes, B.

    2008-01-01

    Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstroms resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

  20. Purification and characterization of a novel GTP-binding protein from human platelet cytosol

    International Nuclear Information System (INIS)

    Human platelet cytosol contains a GTP-binding protein which exhibits a Mr of 37,000 daltons in SDS-PAGE. This protein was purified to apparent homogeneity using conventional and fast protein liquid chromatography. The protein does not exhibit detectable GTPase activity. Analysis of amino acid composition indicates striking analogy to α subunits of previously described membrane bound G-proteins. The purified protein binds GTP or GTPγS specifically with a Kd of 118 nM or 70 nM respectively as determined by using [35S]GTPγS as labeled ligand. GDP or ATP is competitive only at more than 100 fold higher concentrations. Other nucleotides are not competitive at all. The protein at the native state is a dimer of Mr of 74,000 daltons. Binding of GTP is stimulated by various metal ions, dithiothreitol, phospholipids and arachidonic acid, and is inhibited by sulfhydryl inhibitors. Lysophosphatidylcholine appears to be the most potent stimulator. The sensitivity of the GTP binding toward thiol reagents suggests that the cysteinyl residues may be crucial for GTP binding. The functional role of this novel GTP-binding protein remains unknown

  1. Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

    Science.gov (United States)

    Smidt, M P; Russchen, B; Snippe, L; Wijnholds, J; Ab, G

    1995-07-11

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. PMID:7630716

  2. How does a protein reach its binding locus: sliding along DNA chain or not?

    CERN Document Server

    Li, Jingwei

    2016-01-01

    In gene expression, various kinds of proteins need to bind to specific locus of DNA. It is still not clear how these proteins find their target locus. In this study, the mean first-passage time (FPT) of protein binding to its target locus on DNA chain is discussed by a chain-space coupled model. Our results show that the 1-dimensional diffusion constant has a critical value, with which the mean time spent by a protein to find its target locus is almost independent of the binding rate of protein to DNA chain and the detachment rate from DNA chain. Which implies that, the frequency of protein binding to DNA and the sliding time on DNA chain have little influence on the search efficiency, and therefore whether or not the 1-dimensional sliding on DNA chain increases the search efficiency depends on the 1-dimensional diffusion constant of the protein on DNA chain. This study also finds that only protein bindings to DNA loci which are close to the target locus help to increase the search efficiency, while bindings ...

  3. Isolation of Two Strong Poly (U) Binding Proteins from Moderate Halophile Halomonas eurihalina and Their Identification as Cold Shock Proteins

    OpenAIRE

    Usha Kumari Garapati; Tangirala Suryanarayana

    2012-01-01

    Cold shock proteins (Csp) are known to be expressed in response to sudden decrease in temperature. They are thought to be involved in a number of cellular processes viz., RNA chaperone activity, translation, transcription, nucleoid condensation. During our studies on ribosomal protein S1 in moderate halophile Halomonas eurihalina, we observed the presence of two strong poly (U) binding proteins in abundance in cell extracts from cells grown under normal growth conditions. The proteins can be ...

  4. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism).

    OpenAIRE

    Daughaday, W H; Trivedi, B

    1987-01-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, we have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 12...

  5. Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins

    OpenAIRE

    Jönsson, Klas; Guo, Betty P.; Monstein, Hans-Jürg; Mekalanos, John J.; Kronvall, Göran

    2004-01-01

    Helicobacter pylori binds a number of host cell proteins, including the plasma protein plasminogen, which is the proenzyme of the serine protease plasmin. Two H. pylori plasminogen-binding proteins have been described; however, no genes were identified. Here we report the use of a phage display library to clone two genes from the H. pylori CCUG 17874 genome that mediate binding to plasminogen. DNA sequence analysis of one of these genes revealed 96.6% homology with H. pylori 26695 HP0508. A s...

  6. Computational Design of a DNA- and Fc-Binding Fusion Protein

    Directory of Open Access Journals (Sweden)

    Jonas Winkler

    2011-01-01

    Full Text Available Computational design of novel proteins with well-defined functions is an ongoing topic in computational biology. In this work, we generated and optimized a new synthetic fusion protein using an evolutionary approach. The optimization was guided by directed evolution based on hydrophobicity scores, molecular weight, and secondary structure predictions. Several methods were used to refine the models built from the resulting sequences. We have successfully combined two unrelated naturally occurring binding sites, the immunoglobin Fc-binding site of the Z domain and the DNA-binding motif of MyoD bHLH, into a novel stable protein.

  7. Characterisation of the DNA binding domain of the yeast RAP1 protein

    OpenAIRE

    Henry, Y A; Chambers, A.; Tsang, J S; Kingsman, A J; Kingsman, S M

    1990-01-01

    The 827 amino acid yeast RAP1 protein interacts with DNA to regulate gene expression at numerous unrelated loci in the yeast genome. By a combination of amino, carboxy and internal deletions, we have defined an internal 235 amino acid fragment of the yeast RAP1 protein that can bind efficiently to the RAP1 binding site of the PGK Upstream Activation Sequence (UAS). This domain spans residues 361 to 596 of the full length protein and lacks any homology to the DNA binding 'zinc finger' or 'heli...

  8. Multifunctionality and mechanism of ligand binding in a mosquito antiinflammatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Calvo, Eric; Mans, Ben J.; Ribeiro, José M.C.; Andersen, John F.; (NIH)

    2009-04-07

    The mosquito D7 salivary proteins are encoded by a multigene family related to the arthropod odorant-binding protein (OBP) superfamily. Forms having either one or two OBP domains are found in mosquito saliva. Four single-domain and one two-domain D7 proteins from Anopheles gambiae and Aedes aegypti (AeD7), respectively, were shown to bind biogenic amines with high affinity and with a stoichiometry of one ligand per protein molecule. Sequence comparisons indicated that only the C-terminal domain of AeD7 is homologous to the single-domain proteins from A. gambiae, suggesting that the N-terminal domain may bind a different class of ligands. Here, we describe the 3D structure of AeD7 and examine the ligand-binding characteristics of the N- and C-terminal domains. Isothermal titration calorimetry and ligand complex crystal structures show that the N-terminal domain binds cysteinyl leukotrienes (cysLTs) with high affinities (50-60 nM) whereas the C-terminal domain binds biogenic amines. The lipid chain of the cysLT binds in a hydrophobic pocket of the N-terminal domain, whereas binding of norepinephrine leads to an ordering of the C-terminal portion of the C-terminal domain into an alpha-helix that, along with rotations of Arg-176 and Glu-268 side chains, acts to bury the bound ligand.

  9. Parameterization of an effective potential for protein-ligand binding from host-guest affinity data.

    Science.gov (United States)

    Wickstrom, Lauren; Deng, Nanjie; He, Peng; Mentes, Ahmet; Nguyen, Crystal; Gilson, Michael K; Kurtzman, Tom; Gallicchio, Emilio; Levy, Ronald M

    2016-01-01

    Force field accuracy is still one of the "stalemates" in biomolecular modeling. Model systems with high quality experimental data are valuable instruments for the validation and improvement of effective potentials. With respect to protein-ligand binding, organic host-guest complexes have long served as models for both experimental and computational studies because of the abundance of binding affinity data available for such systems. Binding affinity data collected for cyclodextrin (CD) inclusion complexes, a popular model for molecular recognition, is potentially a more reliable resource for tuning energy parameters than hydration free energy measurements. Convergence of binding free energy calculations on CD host-guest systems can also be obtained rapidly, thus offering the opportunity to assess the robustness of these parameters. In this work, we demonstrate how implicit solvent parameters can be developed using binding affinity experimental data and the binding energy distribution analysis method (BEDAM) and validated using the Grid Inhomogeneous Solvation Theory analysis. These new solvation parameters were used to study protein-ligand binding in two drug targets against the HIV-1 virus and improved the agreement between the calculated and the experimental binding affinities. This work illustrates how benchmark sets of high quality experimental binding affinity data and physics-based binding free energy models can be used to evaluate and optimize force fields for protein-ligand systems. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26256816

  10. Novel RNA-binding properties of the MTG chromatin regulatory proteins

    NARCIS (Netherlands)

    S. Rossetti (Stefano); L. van Unen (Leontine); N. Sacchi; A.T. Hoogeveen (Andre)

    2008-01-01

    textabstractBackground: The myeloid translocation gene (MTG) proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the

  11. Transmissible gastroenteritis virus; identification of M protein-binding peptide ligands with antiviral and diagnostic potential

    Science.gov (United States)

    The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and ...

  12. Design and synthesis of ATP-based nucleotide analogues and profiling of nucleotide-binding proteins

    NARCIS (Netherlands)

    Wolters, Justina. C.; Roelfes, Johannes; Poolman, Bert

    2011-01-01

    Two nucleotide-based probes were designed and synthesized in order to enrich samples for specific classes of proteins by affinity-based protein profiling. We focused on the profiling of adenine nucleotide-binding proteins. Two properties were considered in the design of the probes: the bait needs to

  13. Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A.

    Science.gov (United States)

    Maurer, Manuela; de Beer, Stephanie B A; Oostenbrink, Chris

    2016-01-01

    The periplasmic oligopeptide binding protein A (OppA) represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK), but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated) unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data. PMID:27092480

  14. Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin.

    Science.gov (United States)

    Murahashi, Masataka; Simizu, Siro; Morioka, Masahiko; Umezawa, Kazuo

    2016-08-01

    15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity. PMID:27261432

  15. An Overview of the Prediction of Protein DNA-Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

    Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  16. Mechanosensitive kinetic preference of actin-binding protein to actin filament

    Science.gov (United States)

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

    The kinetic preference of actin-binding proteins to actin filaments is altered by external forces on the filament. Such an altered kinetic preference is largely responsible for remodeling the actin cytoskeletal structure in response to intracellular forces. During remodeling, actin-binding proteins and actin filaments interact under isothermal conditions, because the cells are homeostatic. In such a temperature homeostatic state, we can rigorously and thermodynamically link the chemical potential of actin-binding proteins to stresses on the actin filaments. From this relationship, we can construct a physical model that explains the force-dependent kinetic preference of actin-binding proteins to actin filaments. To confirm the model, we have analyzed the mechanosensitive alternation of the kinetic preference of Arp2/3 and cofilin to actin filaments. We show that this model captures the qualitative responses of these actin-binding proteins to the forces, as observed experimentally. Moreover, our theoretical results demonstrate that, depending on the structural parameters of the binding region, actin-binding proteins can show different kinetic responses even to the same mechanical signal tension, in which the double-helix nature of the actin filament also plays a critical role in a stretch-twist coupling of the filament.

  17. Predicting binding affinities of protein ligands from three-dimensional models: application to peptide binding to class I major histocompatibility proteins

    DEFF Research Database (Denmark)

    Rognan, D; Lauemoller, S L; Holm, A; Buus, S; Tschinke, V

    1999-01-01

    A simple and fast free energy scoring function (Fresno) has been developed to predict the binding free energy of peptides to class I major histocompatibility (MHC) proteins. It differs from existing scoring functions mainly by the explicit treatment of ligand desolvation and of unfavorable protein...... interactions were found to contribute the most to HLA-A0201-peptide interactions, whereas H-bonding predominates in H-2K(k) recognition. Both cross-validated models were afterward used to predict the binding affinity of a test set of 26 peptides to HLA-A0204 (an HLA allele closely related to HLA-A0201) and of...

  18. Global analysis of small molecule binding to related protein targets.

    Directory of Open Access Journals (Sweden)

    Felix A Kruger

    2012-01-01

    Full Text Available We report on the integration of pharmacological data and homology information for a large scale analysis of small molecule binding to related targets. Differences in small molecule binding have been assessed for curated pairs of human to rat orthologs and also for recently diverged human paralogs. Our analysis shows that in general, small molecule binding is conserved for pairs of human to rat orthologs. Using statistical tests, we identified a small number of cases where small molecule binding is different between human and rat, some of which had previously been reported in the literature. Knowledge of species specific pharmacology can be advantageous for drug discovery, where rats are frequently used as a model system. For human paralogs, we demonstrate a global correlation between sequence identity and the binding of small molecules with equivalent affinity. Our findings provide an initial general model relating small molecule binding and sequence divergence, containing the foundations for a general model to anticipate and predict within-target-family selectivity.

  19. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel Miotto; Thuesen, Cathrine K; Bøtkjær, Kenneth A;

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless......, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with...... therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to...

  20. The Arabidopsis mitogen-activated protein kinase 6 is associated with γ-tubulin on microtubules, phosphorylates EB1c and maintains spindle orientation under nitrosative stress

    Czech Academy of Sciences Publication Activity Database

    Kohoutová, Lucie; Kourová, Hana; Nagy, S. K.; Volc, Jindřich; Halada, Petr; Mészáros, T.; Meskiene, I.; Bögre, L.; Binarová, Pavla

    2015-01-01

    Roč. 207, č. 4 (2015), s. 1061-1074. ISSN 0028-646X R&D Projects: GA MŠk 7AMB13AT013; GA ČR GAP501/12/2333 Institutional support: RVO:61388971 Keywords : Arabidopsis * cell division * EB1c Subject RIV: EE - Microbiology, Virology Impact factor: 7.672, year: 2014

  1. A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

    Science.gov (United States)

    Safadi, F.; Reddy, V. S.; Reddy, A. S.

    2000-01-01

    Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

  2. Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus

    Science.gov (United States)

    Kotova, E. S.; Akopov, S. B.; Didych, D. A.; Petrova, N. V.; Iarovaia, O. V.; Razin, S. V.; Nikolaev, L. G.

    2016-01-01

    A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently – in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected. PMID:27099788

  3. Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus.

    Science.gov (United States)

    Kotova, E S; Akopov, S B; Didych, D A; Petrova, N V; Iarovaia, O V; Razin, S V; Nikolaev, L G

    2016-01-01

    A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected. PMID:27099788

  4. Arabidopsis Pumilio protein APUM5 suppresses Cucumber mosaic virus infection via direct binding of viral RNAs.

    Science.gov (United States)

    Huh, Sung Un; Kim, Min Jung; Paek, Kyung-Hee

    2013-01-01

    Posttranscriptional/translational regulation of gene expression is mediated by diverse RNA binding proteins and plays an important role in development and defense processes. Among the RNA-binding proteins, the mammalian Pumilio RNA-binding family (Puf) acts as posttranscriptional and translational repressors. An Arabidopsis Puf mutant, apum5-D, was isolated during a T-DNA insertional mutant screen for mutants with reduced susceptibility to Cucumber mosaic virus (CMV) infection. Interestingly, CMV RNA contained putative Pumilio-homology domain binding motifs in its 3' untranslated region (UTR) and internal places in its genome. APUM5 directly bound to the 3' UTR motifs and some internal binding motifs in CMV RNAs in vitro and in vivo. We showed that APUM5 acts as a translational repressor that regulates the 3' UTR of CMV and affects CMV replication. This study uncovered a unique defense system that Arabidopsis APUM5 specifically regulates CMV infection by the direct binding of CMV RNAs. PMID:23269841

  5. TAFII170 Interacts with the Concave Surface of TATA-Binding Protein To Inhibit Its DNA Binding Activity

    Science.gov (United States)

    Pereira, Lloyd A.; van der Knaap, Jan A.; van den Boom, Vincent; van den Heuvel, Fiona A. J.; Timmers, H. T. Marc

    2001-01-01

    The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAFII170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAFII170. We have defined the TBP interaction domain of TAFII170 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 (TBPAS) containing a triple mutation in the concave surface is defective for binding the TAFII170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAFII170 residues 290 to 381 can inhibit the interaction between Drosophila TAFII230 (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 TAFII170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBPAS mutant is less sensitive to TAFII170 inhibition. Collectively, our results support a mechanism in which TAFII170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface. PMID:11585931

  6. A machine learning approach for the identification of odorant binding proteins from sequence-derived properties

    Directory of Open Access Journals (Sweden)

    Suganthan PN

    2007-09-01

    Full Text Available Abstract Background Odorant binding proteins (OBPs are believed to shuttle odorants from the environment to the underlying odorant receptors, for which they could potentially serve as odorant presenters. Although several sequence based search methods have been exploited for protein family prediction, less effort has been devoted to the prediction of OBPs from sequence data and this area is more challenging due to poor sequence identity between these proteins. Results In this paper, we propose a new algorithm that uses Regularized Least Squares Classifier (RLSC in conjunction with multiple physicochemical properties of amino acids to predict odorant-binding proteins. The algorithm was applied to the dataset derived from Pfam and GenDiS database and we obtained overall prediction accuracy of 97.7% (94.5% and 98.4% for positive and negative classes respectively. Conclusion Our study suggests that RLSC is potentially useful for predicting the odorant binding proteins from sequence-derived properties irrespective of sequence similarity. Our method predicts 92.8% of 56 odorant binding proteins non-homologous to any protein in the swissprot database and 97.1% of the 414 independent dataset proteins, suggesting the usefulness of RLSC method for facilitating the prediction of odorant binding proteins from sequence information.

  7. [Electron paramagnetic resonance study of the interactions between steroid hormones and binding proteins].

    Science.gov (United States)

    Basset, M; Chambaz, E M; Defaye, G; Metz, B

    1978-01-01

    Interaction of a spin labeled corticosteroid (desoxycorticosterone nitroxyde: DOC -NO) with three purified proteins (albumin, transcortin, progesterone binding protein: PBG) was studied by electron spin resonance (ESR) spectroscopy. DOC-NO was competitive with natural corticosteroids and therefore bound at the same site to specific binding proteins. ESR spectra in the presence of each of the proteins showed an immobilized (bound) form of the spin labeled steroid and allowed the calculation of the corresponding association constant (Ka) at equilibrium. The three binding proteins could be characterized by the ESR parameters of the DOC-NO bound form. The thermodynamic parameters (deltaH, deltaS) of the steroid-protein interactions were calculated from the ESR data obtained within a wide temperature range (3--40 degrees C). The ESR spectra width (2T) was used to evaluate the polarity of the spin label environment within the steroid binding site: a hydrophobic character was observed for transcortin whereas PBG exhibited a more hydrophilic steroid binding sits. The rotational correlation time of the three protein DOC-NO complexes at equilibrium were calculated from ESR data; the results were correlated with the protein molecular size and suggested a non spherical shape for the binding macromolecule in solution. Spin labelling of biologically active steroids thus provides a novel approach for the study of the interaction of these hormones with their binding protein. Providing a suitable spin label, the ESR parameters may allow the characterization of several types of binding sites of different biological significance for the same hormone, in biological fluids as well as in target tissues. PMID:83166

  8. Knowledge-based annotation of small molecule binding sites in proteins

    Directory of Open Access Journals (Sweden)

    Panchenko Anna R

    2010-07-01

    Full Text Available Abstract Background The study of protein-small molecule interactions is vital for understanding protein function and for practical applications in drug discovery. To benefit from the rapidly increasing structural data, it is essential to improve the tools that enable large scale binding site prediction with greater emphasis on their biological validity. Results We have developed a new method for the annotation of protein-small molecule binding sites, using inference by homology, which allows us to extend annotation onto protein sequences without experimental data available. To ensure biological relevance of binding sites, our method clusters similar binding sites found in homologous protein structures based on their sequence and structure conservation. Binding sites which appear evolutionarily conserved among non-redundant sets of homologous proteins are given higher priority. After binding sites are clustered, position specific score matrices (PSSMs are constructed from the corresponding binding site alignments. Together with other measures, the PSSMs are subsequently used to rank binding sites to assess how well they match the query and to better gauge their biological relevance. The method also facilitates a succinct and informative representation of observed and inferred binding sites from homologs with known three-dimensional structures, thereby providing the means to analyze conservation and diversity of binding modes. Furthermore, the chemical properties of small molecules bound to the inferred binding sites can be used as a starting point in small molecule virtual screening. The method was validated by comparison to other binding site prediction methods and to a collection of manually curated binding site annotations. We show that our method achieves a sensitivity of 72% at predicting biologically relevant binding sites and can accurately discriminate those sites that bind biological small molecules from non-biological ones. Conclusions

  9. Statistical-mechanical lattice models for protein-DNA binding in chromatin

    International Nuclear Information System (INIS)

    Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibria measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical-mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quantitative models for the regulation of gene expression.

  10. Using circular permutation analysis to redefine the R17 coat protein binding site.

    Science.gov (United States)

    Gott, J M; Pan, T; LeCuyer, K A; Uhlenbeck, O C

    1993-12-14

    The bacteriophage R17 coat protein binding site consists of an RNA hairpin with a single purine nucleotide bulge in the helical stem. Circular permutation analysis (CPA) was used to examine binding effects caused by a single break in the phosphodiester backbone. This method revealed that breakage of all but one phosphodiester bond within a well-defined binding site substantially reduced the binding affinity. This is probably due to destabilization of the hairpin structure upon breaking the ribose phosphates at these positions. One circularly permuted isomer with the 5' and 3' ends at the bulged nucleotide bound with wild-type affinity. However, extending the 5' end of this CP isomer greatly reduces binding, making it unlikely that this circularly permuted binding site will be active when embedded in a larger RNA. CPA also locates the 5' and 3' boundaries of protein binding sites on the RNA. The 5' boundary of the R17 coat protein site as defined by CPA was two nucleotides shorter (nucleotides -15 to +2) than the previously determined site (-17 to +2). The smaller binding site was verified by terminal truncation experiments. A minimal-binding fragment (-14 to +2) was synthesized and was found to bind tightly to the coat protein. The site size determined by 3-ethyl-1-nitrosourea-modification interference was larger at the 5' end (-16 to +1), probably due, however, to steric effects of ethylation of phosphate oxygens. Thus, the apparent site size of a protein binding site is dependent upon the method used. PMID:7504949

  11. Developing novel single molecule analyses of the single-stranded DNA binding protein from Sulfolobus solfataricus

    OpenAIRE

    Morten, Michael J.

    2015-01-01

    Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB)...

  12. A computational method for the analysis and prediction of protein:phosphopeptide-binding sites

    OpenAIRE

    Joughin, Brian A.; Tidor, Bruce; Yaffe, Michael B.

    2005-01-01

    Phosphopeptide-binding domains, including the FHA, SH2, WW, WD40, MH2, and Polo-box domains, as well as the 14-3-3 proteins, exert control functions in important processes such as cell growth, division, differentiation, and apoptosis. Structures and mechanisms of phosphopeptide binding are generally diverse, revealing few general principles. A computational method for analysis of phosphopeptide-binding domains was therefore developed to elucidate the physical and chemical nature of phosphopep...

  13. In Vitro Binding Capacity of Bile Acids by Defatted Corn Protein Hydrolysate

    Directory of Open Access Journals (Sweden)

    Pierre Claver Irakoze

    2011-02-01

    Full Text Available Defatted corn protein was digested using five different proteases, Alcalase, Trypsin, Neutrase, Protamex and Flavourzyme, in order to produce bile acid binding peptides. Bile acid binding capacity was analyzed in vitro using peptides from different proteases of defatted corn hydrolysate. Some crystalline bile acids like sodium glycocholate, sodium cholate and sodium deoxycholate were individually tested using HPLC to see which enzymes can release more peptides with high bile acid binding capacity. Peptides from Flavourzyme defatted corn hydrolysate exhibited significantly (p

  14. Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues

    OpenAIRE

    Schulz, Timothy A.; Choi, Mal-Gi; Raychaudhuri, Sumana; Mears, Jason A.; Ghirlando, Rodolfo; Hinshaw, Jenny E.; Prinz, William A.

    2009-01-01

    Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein–related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Usin...

  15. Identification of the Receptor Binding Domain of the Mouse Mammary Tumor Virus Envelope Protein

    OpenAIRE

    Zhang, Yuanming; Rassa, John C.; deObaldia, Maria Elena; Albritton, Lorraine M.; Susan R Ross

    2003-01-01

    Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity...

  16. Selective coactivation of estrogen-dependent transcription by CITED1 CBP/p300-binding protein

    OpenAIRE

    Yahata, Tetsuro; Shao, Wenlin; Endoh, Hideaki; Hur, Jingyung; Coser, Kathryn R.; Sun, Huiping; Ueda, Yoshitaka; Kato, Shigeaki; Isselbacher, Kurt J.; Brown, Myles; Shioda, Toshi

    2001-01-01

    CITED1, a CBP/p300-binding nuclear protein that does not bind directly to DNA, is a transcriptional coregulator. Here, we show evidence that CITED1 functions as a selective coactivator for estrogen-dependent transcription. When transfected, CITED1 enhanced transcriptional activation by the ligand-binding/AF2 domain of both estrogen receptor-α (ERα) and ERβ in an estrogen-dependent manner, but it affected transcriptional activities of other nuclear receptors only marginally. CITED1 bound direc...

  17. Regulation of blood-testis barrier by actin binding proteins and protein kinases.

    Science.gov (United States)

    Li, Nan; Tang, Elizabeth I; Cheng, C Yan

    2016-03-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis, since the onset of meiosis and spermiogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular, at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin-binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  18. Loss of Fatty Acid Binding Protein 4/aP2 Reduces Macrophage Inflammation Through Activation of SIRT3.

    Science.gov (United States)

    Xu, Hongliang; Hertzel, Ann V; Steen, Kaylee A; Bernlohr, David A

    2016-03-01

    Activation of proinflammatory macrophages plays an important role in the pathogenesis of insulin resistance, type 2 diabetes, and atherosclerosis. Previous work using high fat-fed mice has shown that ablation of the adipocyte fatty acid binding protein (FABP4/aP2) in macrophages leads to an antiinflammatory state both in situ and in vivo, and the mechanism is linked, in part, to increased intracellular monounsaturated fatty acids and the up-regulation of uncoupling protein 2. Here, we show that loss of FABP4/aP2 in macrophages additionally induces sirtuin 3 (SIRT3) expression and that monounsaturated fatty acids (C16:1, C18:1) lead to increased SIRT3 protein expression. Increased expression of SirT3 in FABP4/aP2 null macrophages occurs at the protein level with no change in SirT3 mRNA. When compared with controls, silencing of SIRT3 in Raw246.7 macrophages leads to increased expression of inflammatory cytokines, inducible nitric oxide synthase and cyclooxygenase 2. In contrast, loss of SIRT3 in FABP4/aP2-deficient macrophages attenuates the suppressed inflammatory signaling, reduced reactive oxygen species production, lipopolysaccharide-induced mitochondrial dysfunction, and increased fatty acid oxidation. These results suggest that the antiinflammatory phenotype of FABP4/aP2 null mice is mediated by increased intracellular monounsaturated fatty acids leading to the increased expression of both uncoupling protein 2 and SirT3. PMID:26789108

  19. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

    DEFF Research Database (Denmark)

    Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel; Li, Shengting; Fryland, Tue; Børglum, Anders; Bolund, Lars; Nielsen, Anders Lade

    2015-01-01

    Background FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins......IP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression at...... involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins....

  20. Neutrophils and the calcium-binding protein MRP-14 mediate carrageenan-induced antinociception in mice

    Directory of Open Access Journals (Sweden)

    Rosana L. Pagano

    2002-01-01

    Full Text Available Background: We have previously shown that the calcium-binding protein MRP-14 secreted by neutrophils mediates the antinociceptive response in an acute inflammatory model induced by the intraperitoneal injection of glycogen in mice.

  1. Effect of membrane protein concentration on binding of 3H-imipramine in human platelets

    International Nuclear Information System (INIS)

    Binding of 3H-imipramine to platelet membranes has been implicated as a marker for depression. Comparing 3H-IMI binding between depressed patients and normal subjects we observed an increase in the dissociation constant Kd with increasing membrane protein. This phenomenon was studied more rigorously in five normal subjects. Platelet membranes were prepared and adjusted to four concentrations of protein ranging from 100 to 800 micrograms/ml. The 3H-IMI binding parameters of maximum binding sites number (Bmax) and Kd were obtained by Scatchard analysis at each membrane concentration. A positive linear relationship was found between K/sub d/ values and the concentration of membrane protein in the assay, but no change was observed in Bmax. The variability in Kd values reported in the literature may be accounted for in part by the different concentrations of membrane protein used in various studies

  2. Human kidney amiloride-binding protein: cDNA structure and functional expression

    International Nuclear Information System (INIS)

    Phenamil, an analog of amiloride, is a potent blocker of the epithelial Naplus channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Naplus channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein

  3. Identification of a binding protein to the X gene promoter region of hepatitis B virus.

    Science.gov (United States)

    Nakamura, I; Koike, K

    1992-12-01

    The X protein of hepatitis B virus (HBV) is a transactivator to homologous and heterologous viral and cellular transcriptional regulatory elements. One sequence-specific binding protein, whose binding site located from nt 1102 to nt 1117 of HBV DNA, was identified by mobility shift assay and DNase I foot-printing analysis. A CAT assay experiment demonstrated this 16-bp binding site to have a promoter activity in the X gene transcription. The 58-bp DNA fragment (nt 1085 to nt 1142), which contains the above binding site, could be enhanced by the HBV enhancer. Mobility shift assay using the mutated 58-bp DNA fragments as probes, showed that the mutation, which damaged the palindrome structure between nt 1105 and nt 1112, resulted in loss of the binding activity. This mutation also remarkably reduced the promoter activity. The binding site differed from the target sequences of known transcriptional factors. This factor was thus concluded to be a binding protein to the X gene promoter (X-PBP) of HBV. A homology search demonstrated the binding site to be highly homologous to the promoter elements of human laminin receptor (2H5epitope) and lipoprotein receptor-related protein (LRP) genes. PMID:1448911

  4. Draft genome sequences of 18 oral streptococcus strains that encode amylase-binding proteins.

    Science.gov (United States)

    Sabharwal, Amarpreet; Liao, Yu-Chieh; Lin, Hsin-Hung; Haase, Elaine M; Scannapieco, Frank A

    2015-01-01

    A number of commensal oral streptococcal species produce a heterogeneous group of proteins that mediate binding of salivary α-amylase. This interaction likely influences streptococcal colonization of the oral cavity. Here, we present draft genome sequences of several strains of oral streptococcal species that bind human salivary amylase. PMID:25999552

  5. Draft Genome Sequences of 18 Oral Streptococcus Strains That Encode Amylase-Binding Proteins

    OpenAIRE

    Sabharwal, Amarpreet; Liao, Yu-Chieh; Lin, Hsin-Hung; Haase, Elaine M.; Scannapieco, Frank A.

    2015-01-01

    A number of commensal oral streptococcal species produce a heterogeneous group of proteins that mediate binding of salivary α-amylase. This interaction likely influences streptococcal colonization of the oral cavity. Here, we present draft genome sequences of several strains of oral streptococcal species that bind human salivary amylase.

  6. Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

    NARCIS (Netherlands)

    Hollenstein, K.; Comellas-Bigler, M.; Bevers, L.E.; Feiters, M.C.; Meyer-Klaucke, W.; Hagedoorn, P.-L.; Locher, K.P.

    2009-01-01

    Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO4 2−) and tungstate (WO4 2−). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across th

  7. The Effect of Calcium on the Binding of Calmodulin to Calcium/Calmodulin Protein Kinase II.

    Science.gov (United States)

    Porta, Angela R.

    2000-01-01

    Introduces a follow-up laboratory experiment demonstrating the formation change when calcium binds to calmodulin. This conformation change allows this complex to bind to a target protein. Presents the necessary information to conduct the experiment and discusses the results. (YDS)

  8. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

    KAUST Repository

    Wong, Ka-Chun

    2015-06-11

    Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  9. Stepwise bending of DNA by a single TATA box binding protein

    DEFF Research Database (Denmark)

    Tolic-Nørrelykke, Simon F; Rasmussen, Mette B; Pavone, Francesco S; Berg-Sørensen, Kirstine; Oddershede, Lene B.

    2006-01-01

    The TATA-box binding protein (TBP) is required by all three eucaryotic RNA polymerases for the initiation of transcription from most promoters. TBP recognizes, binds to, and bends promoter sequences called "TATA-boxes" in the DNA. We present results from the study of individual Saccharomyces cere...

  10. Isolation and characterization of porcine mannan-binding proteins of different size and ultrastructure

    DEFF Research Database (Denmark)

    Storgaard, P; Nielsen, EH; Andersen, Ove;

    1996-01-01

    mouse and rat MBP-C (41-45% identity). Both pMBPs exhibited Ca2+-dependent binding to D-mannose immobilized on agarose but no significant binding to N-acetyl-D-glucosamine- or fucose-agarose. The results further suggested the presence of a third pMBP which copurified with pMBP-27 but this protein was...

  11. Discovering protein-DNA binding sequence patterns using association rule mining.

    Science.gov (United States)

    Leung, Kwong-Sak; Wong, Ka-Chun; Chan, Tak-Ming; Wong, Man-Hon; Lee, Kin-Hong; Lau, Chi-Kong; Tsui, Stephen K W

    2010-10-01

    Protein-DNA bindings between transcription factors (TFs) and transcription factor binding sites (TFBSs) play an essential role in transcriptional regulation. Over the past decades, significant efforts have been made to study the principles for protein-DNA bindings. However, it is considered that there are no simple one-to-one rules between amino acids and nucleotides. Many methods impose complicated features beyond sequence patterns. Protein-DNA bindings are formed from associated amino acid and nucleotide sequence pairs, which determine many functional characteristics. Therefore, it is desirable to investigate associated sequence patterns between TFs and TFBSs. With increasing computational power, availability of massive experimental databases on DNA and proteins, and mature data mining techniques, we propose a framework to discover associated TF-TFBS binding sequence patterns in the most explicit and interpretable form from TRANSFAC. The framework is based on association rule mining with Apriori algorithm. The patterns found are evaluated by quantitative measurements at several levels on TRANSFAC. With further independent verifications from literatures, Protein Data Bank and homology modeling, there are strong evidences that the patterns discovered reveal real TF-TFBS bindings across different TFs and TFBSs, which can drive for further knowledge to better understand TF-TFBS bindings. PMID:20529874

  12. SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

    Science.gov (United States)

    Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

    2016-04-01

    The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. PMID:27052731

  13. Protein 90 Recognized as an Iron-Binding Protein Associated with the Plasma Membrane of HeLa Cells

    Czech Academy of Sciences Publication Activity Database

    Kovář, Jan; Štýbrová, Hana; Novák, J.; Ehrlichová, Marie; Truksa, Jaroslav; Koc, Michal; Kriegerbecková, Karin; Scheiber-Mojdehkar, B.; Goldenberg, H.

    1-2, č. 14 (2004), s. 41-46. ISSN 1015-8987 R&D Projects: GA AV ČR IAA5052702; GA ČR GA301/01/0041 Keywords : heat shock protein 90 * iron - binding protein * plasma membrane Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.093, year: 2004

  14. Outer membrane protein binding sites of complement component 3 during opsonization of Haemophilus influenzae.

    OpenAIRE

    Hetherington, S V; Patrick, C C; Hansen, E J

    1993-01-01

    Complement component 3 (C3) binding to Haemophilus influenzae type b (Hib) is an important step in host defense against invasive disease, but the details of this process remain poorly understood. We have shown that the P1 and P2 outer membrane proteins (OMPs) serve as binding sites for C3 on serum-opsonized Hib. Whole-cell lysates of opsonized Hib were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose. Immunobl...

  15. Characterization of major recognition sequences for a herpes simplex virus type 1 origin-binding protein.

    OpenAIRE

    Koff, A; Tegtmeyer, P

    1988-01-01

    To investigate early initiation events in the replication of herpes simplex virus type 1, we analyzed interactions of proteins from infected cell extracts with the small origin of herpes simplex virus type 1 (oris1). Using the mobility shift assay, we detected two origin-specific binding interactions. We characterized the more prominent interaction on both strands of the DNA duplex with DNase I protection and methylation interference assays. Protein binding protects 17 bases of DNA on each st...

  16. Plasma levels of osteocalcin and retinol binding protein-4 in patients with medullary thyroid carcinoma

    Directory of Open Access Journals (Sweden)

    Jabar Lotfi

    2014-04-01

    Conclusion: According to difference between plasma levels of osteocalcin and retinol binding protein-4 in patients suffered of medullary thyroid carcinoma comparison with normal subjects, it can be said that, probably medullary thyroid carcinoma has effect on bone and adipose tissue metabolism, so osteocalcin and retinol binding protein-4 hormones have potential to be used for confirmation of diagnosis or following treatment of medullary thyroid carcinoma.

  17. APUM5, encoding a Pumilio RNA binding protein, negatively regulates abiotic stress responsive gene expression

    OpenAIRE

    Huh, Sung Un; Paek, Kyung-Hee

    2014-01-01

    Background A mutant screening was carried out previously to look for new genes related to the Cucumber mosaic virus infection response in Arabidopsis. A Pumilio RNA binding protein-coding gene, Arabidopsis Pumilio RNA binding protein 5 (APUM5), was obtained from this screening. Results APUM5 transcriptional profiling was carried out using a bioinformatics tool. We found that APUM5 was associated with both biotic and abiotic stress responses. However, bacterial and fungal pathogen infection su...

  18. Crystallographic studies on B12 binding proteins in eukaryotes and prokaryotes

    OpenAIRE

    Sukumar, Narayanasami

    2013-01-01

    The x-ray crystal structures of several important vitamin B12 binding proteins that have been solved in recent years have enhanced our current understanding in the vitamin B12 field. These structurally diverse groups of B12 binding proteins perform various important biological activities, both by transporting B12 as well as catalyzing various biological reactions. An in-depth comparative analysis of these structures was carried out using PDB coordinates of a carefully chosen database of B12 b...

  19. The Membrane Receptor for Plasma Retinol Binding Protein, a New Type of Cell-Surface Receptor

    OpenAIRE

    Sun, Hui; KAWAGUCHI, RIKI

    2011-01-01

    Vitamin A is essential for diverse aspects of life ranging from embryogenesis to the proper functioning of most adult organs. Its derivatives (retinoid) have potent biological activities such as regulating cell growth and differentiation. Plasma retinol binding protein (RBP) is the specific vitamin A carrier protein in the blood that binds to vitamin A with high affinity and delivers it to target organs. A large amount of evidence has accumulated over the past decades supporting the existence...

  20. Detection and properties of A-factor-binding protein from Streptomyces griseus.

    OpenAIRE

    Miyake, K; Horinouchi, S; Yoshida, M; Chiba, N; Mori, K; Nogawa, N; Morikawa, N; Beppu, T

    1989-01-01

    The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel f...

  1. Predicting the Impact of Missense Mutations on Protein–Protein Binding Affinity

    OpenAIRE

    Li, Minghui; Petukh, Marharyta; Alexov, Emil; Panchenko, Anna R

    2014-01-01

    The crucial prerequisite for proper biological function is the protein’s ability to establish highly selective interactions with macromolecular partners. A missense mutation that alters the protein binding affinity may cause significant perturbations or complete abolishment of the function, potentially leading to diseases. The availability of computational methods to evaluate the impact of mutations on protein–protein binding is critical for a wide range of biomedical applications. Here, we r...

  2. Human TFDP3, a novel DP protein, inhibits DNA binding and transactivation by E2F

    DEFF Research Database (Denmark)

    Qiao, Huan; Di Stefano, Luisa; Tian, Chan;

    2006-01-01

    The two known DP proteins, TFDP1 and -2, bind E2Fs to form heterodimers essential for high affinity DNA binding and efficient transcriptional activation/repression. Here we report the identification of a new member of the DP family, human TFDP3. Despite the high degree of sequence similarity, TFDP3...... a new DP protein and a novel mechanism whereby E2F function is regulated....

  3. In vitro protein binding of liraglutide in human plasma determined by reiterated stepwise equilibrium dialysis

    OpenAIRE

    Plum, Anne; Jensen, Lisbeth Bjerring; Kristensen, Jesper Bøggild

    2013-01-01

    Liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue approved for the treatment of type 2 diabetes. It is based on human GLP-1 with the addition of a 16-carbon fatty acid, which facilitates binding to plasma proteins, thus prolonging the elimination half-life and allowing once-daily administration. It has not been possible to quantify liraglutide protein binding by ultrafiltration (the usual method of choice), as the lipophilic molecule becomes trapped in the filter membrane. There...

  4. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring

    OpenAIRE

    1990-01-01

    Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is...

  5. Separate Respiratory Phenotypes in Methyl-CpG-Binding Protein 2 (Mecp2) Deficient Mice

    OpenAIRE

    Bissonnette, John M.; Knopp, Sharon J.

    2006-01-01

    Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2) that encodes a DNA binding protein involved in gene silencing. Selective deletion of Mecp2 in post-mitotic neurons in mice results in a Rettlike phenotype characterized by disturbances in motor activity and body weight, suggesting that these symptoms are exclusively caused by neuronal deficiency. Included in the RTT phenotype are episodes of respiratory depression...

  6. Prediction of heme binding residues from protein sequences with integrative sequence profiles

    OpenAIRE

    2012-01-01

    Background The heme-protein interactions are essential for various biological processes such as electron transfer, catalysis, signal transduction and the control of gene expression. The knowledge of heme binding residues can provide crucial clues to understand these activities and aid in functional annotation, however, insufficient work has been done on the research of heme binding residues from protein sequence information. Methods We propose a sequence-based approach for accurate prediction...

  7. Coupled dynamics of DNA-breathing and single-stranded DNA binding proteins

    OpenAIRE

    Ambjornsson, Tobias; Metzler, Ralf

    2004-01-01

    We study the size fluctuations of a local denaturation zone in a DNA molecule in the presence of proteins that selectively bind to single-stranded DNA, based on a (2+1)-dimensional master equation. By tuning the physical parameters we can drive the system from undisturbed bubble fluctuations to full, binding protein-induced denaturation. We determine the effective free energy landscape of the DNA-bubble and explore its relaxation modes.

  8. Staphylococcus aureus and Micrococcus luteus peptidoglycan transglycosylases that are not penicillin-binding proteins.

    OpenAIRE

    Park, W; Matsuhashi, M

    1984-01-01

    Major peptidoglycan transglycosylase activities, which synthesize uncross-linked peptidoglycan from lipid-linked precursors, were solubilized from the membranes of Staphylococcus aureus and Micrococcus luteus and were partially purified. The transglycosylase activities were separated from penicillin-binding proteins by solubilization and by purification steps. Therefore, we concluded that these activities were not activities of the penicillin-binding proteins, which are the presumptive peptid...

  9. Calcium binding protein-mediated regulation of voltage-gated calcium channels linked to human diseases

    Institute of Scientific and Technical Information of China (English)

    Nasrin NFJATBAKHSH; Zhong-ping FENG

    2011-01-01

    Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases.

  10. Hormone response element binding proteins: novel regulators of vitamin D and estrogen signaling

    OpenAIRE

    Lisse, Thomas S.; Hewison, Martin; Adams, John S.

    2011-01-01

    Insights from vitamin D-resistant New World primates and their human homologues as models of natural and pathological insensitivity to sterol/steroid action have uncovered a family of novel intracellular vitamin D and estrogen regulatory proteins involved in hormone action. The proteins, known as “vitamin D or estrogen response element-binding proteins”, behave as potent cis-acting, transdominant regulators to inhibit steroid receptor binding to DNA response elements and is responsible for vi...

  11. Electrical Detection of TATA Binding Protein at DNA-Modified Microelectrodes

    OpenAIRE

    Gorodetsky, Alon A.; Ebrahim, Ali; Barton, Jacqueline K.

    2008-01-01

    A simple method for the electrochemical detection of TATA-binding protein is demonstrated at DNA-modified microelectrodes. The assay is general and based on the interruption of DNA-mediated charge transport to Nile Blue, a redox-active probe covalently attached to the DNA base pair stack. Nanomolar quantities of TATA binding protein can be detected on the microelectrodes even in the presence of micromolar amounts of bovine serum albumin, EndonucleaseIII, or Bam HI methyltransferase. The schem...

  12. Biochemical characterization and bacterial expression of an odorant-binding protein from Locusta migratoria.

    Science.gov (United States)

    Ban, L; Scaloni, A; D'Ambrosio, C; Zhang, L; Yahn, Y; Pelosi, P

    2003-02-01

    Analysis of soluble proteins from different body parts of Locusta migratoria revealed a fast-migrating component in native electrophoresis, unique to antennae of both sexes. N-terminal sequence analysis and cloning identified this protein as a member of the insect odorant-binding proteins, carrying a well-conserved six-cysteine motif. Mass spectrometry analysis confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues are paired in an interlocked fashion. The protein was expressed in a bacterial system with yields of about 10 mg/l of culture, mostly present as inclusion bodies. However, this recombinant product was solubilized after disulfide reduction. Air oxidation yielded a species with all disulfides spontaneously formed as in the native counterpart. Both native and recombinant proteins migrated as a dimer in gel filtration chromatography. Ligand binding was measured, using N-phenyl-1-naphthylamine as the fluorescent probe; the affinity of other ligands was measured in competitive binding assays. The protein exhibited great resistance to thermal denaturation even following prolonged treatment at 100 degrees C. A structural model for this dimeric species was generated on the basis of its sequence homology with Bombyx mori pheromone-binding protein, whose three-dimensional structure has been resolved as an unbound species and in complex with its physiological ligand. This is the first report of an odorant-binding protein identified and characterized from Orthoptera. PMID:12678502

  13. Self-Assembly of Protein Monolayers Engineered for Improved Monoclonal Immunoglobulin G Binding

    Directory of Open Access Journals (Sweden)

    Jeremy H. Lakey

    2011-08-01

    Full Text Available Bacterial outer membrane proteins, along with a filling lipid molecule can be modified to form stable self-assembled monolayers on gold. The transmembrane domain of Escherichia coli outer membrane protein A has been engineered to create a scaffold protein to which functional motifs can be fused. In earlier work we described the assembly and structure of an antibody-binding array where the Z domain of Staphylococcus aureus protein A was fused to the scaffold protein. Whilst the binding of rabbit polyclonal immunoglobulin G (IgG to the array is very strong, mouse monoclonal IgG dissociates from the array easily. This is a problem since many immunodiagnostic tests rely upon the use of mouse monoclonal antibodies. Here we describe a strategy to develop an antibody-binding array that will bind mouse monoclonal IgG with lowered dissociation from the array. A novel protein consisting of the scaffold protein fused to two pairs of Z domains separated by a long flexible linker was manufactured. Using surface plasmon resonance the self-assembly of the new protein on gold and the improved binding of mouse monoclonal IgG were demonstrated.

  14. MULTIPLE G PROTEINS COMPETE FOR BINDING WITH THE HUMAN GONADOTROPIN RELEASING HORMONE RECEPTOR

    OpenAIRE

    Knollman, Paul E.; Conn, P. Michael

    2008-01-01

    The GnRH receptor is coupled to G proteins of the families Gq and G11. Gq and G11. Coupling leads to intracellular signaling through the phospholipase C pathway. GnRHR coupling to other G proteins is controversial. This study provides evidence that G protein families Gs, Gi, Gq and G11 complete for binding with the GnRHR. We quantified interactions of over-expressed G proteins with GnRHR by a competitive binding approach, using measurements of second messengers, IP and cAMP. Transient co-tran...

  15. Cloning and characterization of a human c-myc promoter-binding protein.

    OpenAIRE

    Ray, R; Miller, D M

    1991-01-01

    A human cDNA clone encoding a c-myc promoter-binding protein was detected by screening a HeLa cell lambda phage expression cDNA library. The library was screened by using an XhoI-NaeI human c-myc P2 promoter fragment as a probe. The recombinant phage encoded a fusion protein, myc-binding protein 1 (MBP-1), which had an apparent molecular size of 40 kDa. A corresponding protein with a molecular size of 35 kDa was present in a HeLa cell extract. Sequence analysis of the cloned gene reveals an o...

  16. DNA Binding Proteins of the Filamentous Phages CTXφ and VGJφ of Vibrio cholerae▿

    OpenAIRE

    Falero, Alina; Caballero, Andy; Ferrán, Beatriz; Izquierdo, Yovanny; Fando, Rafael; Campos, Javier

    2009-01-01

    The native product of open reading frame 112 (orf112) and a recombinant variant of the RstB protein, encoded by Vibrio cholerae pathogen-specific bacteriophages VGJφ and CTXφ, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJφ; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirem...

  17. The solution structure of the pentatricopeptide repeat protein PPR10 upon binding atpH RNA

    OpenAIRE

    Gully, Benjamin S.; Cowieson, Nathan; Stanley, Will A; Shearston, Kate; Small, Ian D.; Barkan, Alice; Bond, Charles S.

    2015-01-01

    The pentatricopeptide repeat (PPR) protein family is a large family of RNA-binding proteins that is characterized by tandem arrays of a degenerate 35-amino-acid motif which form an α-solenoid structure. PPR proteins influence the editing, splicing, translation and stability of specific RNAs in mitochondria and chloroplasts. Zea mays PPR10 is amongst the best studied PPR proteins, where sequence-specific binding to two RNA transcripts, atpH and psaJ, has been demonstrated to follow a recogniti...

  18. Inhibition of p53 DNA binding by human papillomavirus E6 proteins.

    OpenAIRE

    Lechner, M S; Laimins, L A

    1994-01-01

    Transformation by the human papillomavirus (HPV) early gene products, E6 and E7, involves their interaction with cellular proteins p53 and Rb. Using glutathione S-transferase (GST) fusion proteins, we found that HPV E6 bound human p53 and that the relative efficiency of binding varied such that the GST-HPV type 16 E6 (16E6) protein bound p53 with highest affinity, followed by GST-31E6, GST-18E6, and GST-11E6. The GST-E6 fusion proteins were sufficient for binding p53 purified from a baculovir...

  19. Expression, purification and characterization of methyl DNA binding protein from Bombyx mori

    OpenAIRE

    Uno, Tomohide; Nomura, Yuka; Nakamura, Masahiko; Nakao, Atsushi; Tajima, Shoji; Kanamaru, Kengo; Yamagata, Hiroshi; Iwanaga, Yousuke

    2005-01-01

    A cDNA clone encoding methyl DNA binding domain-containing protein (bMBD2/3) was obtained by homology searches using a Bombyx mori fat body cDNA library. The cDNA encoded a polypeptide with 249 amino acids sharing 54% similarity with the methyl DNA binding protein from Drosophila melanogaster. To characterize the biochemical properties of bMBD2/3, the clone was expressed in Escherichia coli as His-tagged protein. The recombinant protein was purified to homogeneity using Ni-NTA superflow resin...

  20. Isolation and characterization of a new zinc-binding protein from albacore tuna plasma.

    Science.gov (United States)

    Dyke, B; Hegenauer, J; Saltman, P; Laurs, R M

    1987-06-01

    The protein responsible for sequestering high levels of zinc in the plasma of the albacore tuna (Thunnus alalunga) has been isolated by sequential chromatography. The glycoprotein has a molecular weight of 66,000. Approximately 8.2% of its amino acid residues are histidines. Equilibrium dialysis experiments show it to bind 3 mol of zinc/mol of protein. The stoichiometric constant for the association of zinc with a binding site containing three histidines was determined to be 10(9.4). This protein is different from albumin and represents a previously uncharacterized zinc transport protein. PMID:3607021