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Sample records for kringle 4-binding protein

  1. New class of blue animal pigments based on Frizzled and Kringle protein domains.

    PubMed

    Bulina, Maria E; Lukyanov, Konstantin A; Yampolsky, Ilia V; Chudakov, Dmitry M; Staroverov, Dmitry B; Shcheglov, Alexander S; Gurskaya, Nadya G; Lukyanov, Sergey

    2004-10-15

    The nature of coloration in many marine animals remains poorly investigated. Here we studied the blue pigment of a scyfoid jellyfish Rhizostoma pulmo and determined it to be a soluble extracellular 30-kDa chromoprotein with a complex absorption spectrum peaking at 420, 588, and 624 nm. Furthermore, we cloned the corresponding cDNA and confirmed its identity by immunoblotting and mass spectrometry experiments. The chromoprotein, named rpulFKz1, consists of two domains, a Frizzled cysteine-rich domain and a Kringle domain, inserted into one another. Generally, Frizzleds are members of a basic Wnt signal transduction pathway investigated intensely with regard to development and cancerogenesis. Kringles are autonomous structural domains found throughout the blood clotting and fibrinolytic proteins. Neither Frizzled and Kringle domains association with any type of coloration nor Kringle intrusion into Frizzled sequence was ever observed. Thus, rpulFKz1 represents a new class of animal pigments, whose chromogenic group remains undetermined. The striking homology between a chromoprotein and members of the signal transduction pathway provides a novel node in the evolution track of growth factor-mediated morphogenesis compounds. PMID:15297465

  2. NMR solution structure of the neurotrypsin Kringle domain.

    PubMed

    Ozhogina, Olga A; Grishaev, Alexander; Bominaar, Emile L; Patthy, László; Trexler, Maria; Llinás, Miguel

    2008-11-25

    Neurotrypsin is a multidomain protein that serves as a brain-specific serine protease. Here we report the NMR structure of its kringle domain, NT/K. The data analysis was performed with the BACUS (Bayesian analysis of coupled unassigned spins) algorithm. This study presents the first application of BACUS to the structure determination of a 13C unenriched protein for which no prior experimental 3D structure was available. NT/K adopts the kringle fold, consisting of an antiparallel beta-sheet bridged by an overlapping pair of disulfides. The structure reveals the presence of a surface-exposed left-handed polyproline II helix that is closely packed to the core beta-structure. This feature distinguishes NT/K from other members of the kringle fold and points toward a novel functional role for a kringle domain. Functional divergence among kringle domains is discussed on the basis of their surface and electrostatic characteristics. PMID:18956887

  3. Surface proteins of C6/36 cells involved in dengue virus 4 binding and entry.

    PubMed

    Vega-Almeida, Tania Olivia; Salas-Benito, Mariana; De Nova-Ocampo, Mónica Ascensión; Del Angel, Rosa María; Salas-Benito, Juan Santiago

    2013-06-01

    Dengue virus (DENV) is the causative agent of the most important mosquito-borne viral disease, which is endemic to over 100 countries in tropical and subtropical areas of the world. It is transmitted to humans by Aedes mosquitoes. The first step in the viral infection of host cells is virion attachment to the plasma membrane, which is mediated by specific surface molecules. There are several molecules that participate in DENV infection of mosquitoes, but only a few have been identified. In this work, we co-purified 4 proteins from C6/36 cells using a recombinant DENV 4 E protein and identified them as 70 kDa Heat Shock and 70 kDa Heat Shock cognate proteins (HSP70/HSc70), Binding immunoglobulin protein (BiP), Thioredoxin/protein disulphide isomerase (PDI), and 44 kDa Endoplasmic reticulum resident protein (ERp44) via matrix-assisted laser desorption/ionisation time of flight (Maldi-ToF) analysis. Using immunofluorescence and flow cytometry assays, we observed re-localisation of HSP70/HSc70 and, to a lesser extent, BiP to the plasma membrane under stress conditions, such as during DENV infection. By performing binding and infection assays independently, we found that all 4 proteins participate in both processes, but to differing extents: HSP70/HSc70 is the most critical component, while ERp44 is less important. Viral infection was not inhibited when the cells were incubated with antibodies against all of the surface proteins after virus binding, which suggests that DENV entry to C6/36 cells is mediated by these proteins at the same step and not sequentially. PMID:23344777

  4. Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process.

    PubMed

    Leykauf, Kerstin; Salek, Mojibrahman; Bomke, Jörg; Frech, Matthias; Lehmann, Wolf-Dieter; Dürst, Matthias; Alonso, Angel

    2006-09-01

    Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization. PMID:16931598

  5. Regulation of CDK4 activity by a novel CDK4-binding protein, p34SEI-1

    PubMed Central

    Sugimoto, Masataka; Nakamura, Takeshi; Ohtani, Naoko; Hampson, Lynne; Hampson, Ian N.; Shimamoto, Akira; Furuichi, Yasuhiro; Okumura, Ko; Niwa, Shinichiro; Taya, Yoichi; Hara, Eiji

    1999-01-01

    The p16INK4a tumor suppressor inhibits cyclin-dependent kinases (CDK4 and CDK6). Here we report the isolation of a novel gene, SEI-1, whose product (p34SEI-1) appears to antagonize the function of p16INK4a. Addition of p34SEI-1 to cyclin D1–CDK4 renders the complex resistant to inhibition by p16INK4a. Expression of SEI-1 is rapidly induced on addition of serum to quiescent fibroblasts, and ectopic expression of p34SEI-1 enables fibroblasts to proliferate even in low serum concentrations. p34SEI-1 seems to act as a growth factor sensor and may facilitate the formation and activation of cyclin D–CDK complexes in the face of inhibitory levels of INK4 proteins. PMID:10580009

  6. The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch.

    PubMed

    Oberst, Andrew; Malatesta, Martina; Aqeilan, Rami I; Rossi, Mario; Salomoni, Paolo; Murillas, Rodolfo; Sharma, Prashant; Kuehn, Michael R; Oren, Moshe; Croce, Carlo M; Bernassola, Francesca; Melino, Gerry

    2007-07-01

    Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin-protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73 alpha, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73 alpha for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73 alpha and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1(-/-) cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73 alpha and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy. PMID:17592138

  7. Characterization of the kringle fold and identification of a ubiquitous new class of disulfide rotamers.

    PubMed

    Ozhogina, Olga A; Bominaar, Emile L

    2009-11-01

    The disulfide-bridged chains in the kringle (K) and fibronectin type II (FN2) domains are characterized using a taxonomy that considers the regularities in both beta-secondary structure and cystine cluster. The structural core of the kringle fold comprises an assembly of two beta-hairpins (a "beta-meander") accommodating two overlapping disulfides; one cystine is incorporated in adjacent beta-strands, whereas the other is located just beyond the ends of non-adjacent beta-strands. The dispositions of the (N, C) termini of the two overlapping disulfides in the kringle fold are given as (m, j+1) and (i-1, k+1), in which m, i, j, and k (mkringle fold and is referred to as the "disulfide kringle-cross". The metrics of this motif are quantified, revealing structural differences between the two families of the kringle fold. The conformations of disulfides in the kringle fold are poorly accommodated by existing classification schemes. To elucidate the nature of these rotamers we have performed density functional theory (DFT) calculations for diethyl disulfide. A new classification for the disulfide conformations in proteins is proposed, consisting of six rotamer types: spiral, trans-spiral, corner, trans, hook, and staple. Its relation with previous classification schemes is specified. A survey of high-resolution X-ray structures reveals that the disulfide conformations are clustered around the averaged conformations for the six classes. Average conformation dihedral and distance values are in excellent agreement with the DFT values. The two overlapping disulfides in kringle domains adopt the trans-spiral conformation that appears to be ubiquitous (~17%) in proteins. One of the disulfides stretches across the beta

  8. Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen.

    PubMed

    Matsuka, Y V; Novokhatny, V V; Kudinov, S A

    1990-05-31

    The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6-aminohexanoic acid shows that this fragment, besides the high-affinity lysine-binding site with Kd = 2.9 microM, contains two additional lysine-binding sites which differ in binding strength (Kd = 28 microM and Kd = 220 microM). This strongly suggests the existence of a lysine-binding site in each of the first three kringles. 6-Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6-aminohexanoicacid, pentylamine and arginine (Kd values are 3.2 microM, 4.8 microM and 4.3 microM, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine-binding site of this kringle. PMID:2163837

  9. Isolation of C4-binding protein from guinea pig plasma and demonstration of its function as a control protein of the classical complement pathway C3 convertase.

    PubMed

    Burge, J; Nicholson-Weller, A; Austen, K F

    1981-01-01

    A decay-accelerating factor of the classical complement pathway C3 convertase, C4b,2a, has been purified to homogeneity from guinea pig plasma by a 5-step procedure that includes 5% polyethyleneglycol-4000 (PEG-4000) precipitation, Sepharose 6B gel filtration, heparin-Sepharose chromatography, DE-52 anion exchange chromatography, and Sepharose-C4gp affinity chromatography. The protein elicited a monospecific antiserum in a rabbit and was found with the Mancini technique in both normal and C4-deficient guinea pig plasma at a concentration of 60 microgram/ml. The purified protein gave a single stained band of 550,000 m.w. on SDS-PAGE under nonreducing conditions and a single band of 72,000 m.w. with reduction and alkylation. On the basis of its m.w. and subunit structure, ability to bind to a C4 affinity column, and ability to regulate the classical C system by accelerating the decay of the classical C3 convertase this protein represents the guinea pig analog of the human C4-binding protein. PMID:6778916

  10. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    SciTech Connect

    Chen, Weizao; Feng, Yang; Wang, Yanping; Zhu, Zhongyu; Dimitrov, Dimiter S.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  11. Assessment of IgE and IgG4 Binding Capacities of Cow's Milk Proteins Selectively Altered by Proteases.

    PubMed

    Charcosset, Alexandre; Adel-Patient, Karine; Dupont, Christophe; Bernard, Hervé

    2016-05-01

    Specific IgE and IgG4 have been reported to play key roles in the context of IgE-mediated cow's milk allergy (CMA), but the persistence of their epitopes in milk hydrolysates has not been evaluated. Using sera from 32 CMA patients, 6 CMA patients treated by epicutaneous therapy (CM-treated), and 4 CM-tolerant peanut allergic patients, we analyzed the IgE and IgG4 binding capacities related to major milk allergens in processed milk. Different proteases (plasmin, chymosin, α-chymotrypsin, or pepsin) were used progressively and selectively to hydrolyze β-lactoglobulin (β-LG) and casein (CN) in milk. We then showed that proteases differentially affect IgE or IgG4 immunoreactivities of CN and β-LG and also that we could not relate IgE and/or IgG4 levels or specificities to milk hydrolysates to the clinical status of the patients. PMID:27015440

  12. Prothrombin kringle-2 domain has a growth inhibitory activity against basic fibroblast growth factor-stimulated capillary endothelial cells.

    PubMed

    Lee, T H; Rhim, T; Kim, S S

    1998-10-30

    Recently, O'Reilly et al. (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315-328; O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997) Cell 88, 277-285) developed a simple in vitro angiogenesis assay system using bovine capillary endothelial cell proliferation and purified potent angiogenic inhibitors, including angiostatin and endostatin. Using a simple in vitro assay for angiogenesis, we purified a protein molecule that showed anti-endothelial cell proliferative activity from the serum of New Zealand White rabbits, which was stimulated by lipopolysaccharide. The purified protein showed only bovine capillary endothelial cell growth inhibition and not any cytotoxicity. This molecule was identified as a prothrombin kringle-2 domain (fragment-2) using Edman degradation and the amino acid sequence deduced from the cloned cDNA. Both the prothrombin kringle-2 domain released from prothrombin by factor Xa cleavage and the angiogenic inhibitor purified from rabbit sera exhibited anti-endothelial cell proliferative activity. The recombinant rabbit prothrombin kringle-2 domain showed potent inhibitory activity with half-maximal concentrations (ED50) of 2 microg/ml media. As in angiostatin, the recombinant rabbit prothrombin kringle-2 domain also inhibited angiogenesis in the chorioallantoic membrane of chick embryos. PMID:9786880

  13. The genes encoding for D4Z4 binding proteins HMGB2, YY1, NCL, and MYOD1 are excluded as candidate genes for FSHD1B.

    PubMed

    Bastress, K L; Stajich, J M; Speer, M C; Gilbert, J R

    2005-04-01

    Facioscapulohumeral muscular dystrophy is a disease of skeletal muscle, with symptoms including both facial and shoulder girdle weakness and progression to involve the pelvic girdle and extremities in the majority of cases. For most cases of FSHD, the molecular basis of the disease can be identified as a partial deletion of the D4Z4 repeat array on the end of the long arm of chromosome 4. However, in up to 5% of FSHD families there is no linkage to 4q35. These cases are designated as FSHD1B. Proteins have been identified that bind to the D4Z4 repeats of chromosome 4q35. The genes encoding D4Z4 binding proteins YY1, HMGB2, NCL, and MYOD1 were investigated as candidate genes for FSHD1B. Coding sequences and promoter region were analyzed for HMBG2 and no sequence variations were detected. For YY1, all five exons were analyzed and a polymorphism was detected in both the unaffected and affected populations. In nucleolin (NCL), several SNPs were identified, including a SNP causing the non-synonymous change P515H; however, all polymorphisms either occurred in control samples or were previously reported. A novel polymorphism was also detected in MYOD1, but did not represent a disease-specific variation. These results suggest that HMBG2, YY1, NCL, and MYOD1 are unlikely to represent the genes responsible for FSHD in these families. PMID:15792872

  14. A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice

    PubMed Central

    Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J.

    2016-01-01

    DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans. PMID:27358023

  15. A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice.

    PubMed

    Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J

    2016-01-01

    DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans. PMID:27358023

  16. High-level production of Fc-fused kringle domain in Pichia pastoris.

    PubMed

    Jeong, Gu Min; Lee, Yong Jae; Kim, Yong Sung; Jeong, Ki Jun

    2014-06-01

    Recently, as a new non-immunoglobulin-based protein scaffold, a human kringle domain was successfully engineered toward biologically functional agonists and antagonists. In this study, the fed-batch cultivation conditions were optimized for enhanced production of an Fc-fused kringle domain (KD548-Fc) in Pichia pastoris. Fed-batch cultivations were performed in 5-l laboratory-scale bioreactors, and in order to find the optimal conditions for high-level production of KD548-Fc, several parameters including the initial carbon source (glycerol) concentration, temperature, and pH were investigated. When cells were cultivated at pH 4.0 and 25 °C with 9.5 % glycerol in the initial medium, the highest production yield (635 mg/l) was achieved with high productivity (7.2 mg/l/h). Furthermore, functional KD548-Fc was successfully purified from the culture broth using a simple purification procedure with high purity and recovery yield. PMID:24682857

  17. High level expression of kringle 5 fragment of plasminogen in Pichia pastoris.

    PubMed

    Zhou, Yufei; Zheng, Quan; Gao, Jin; Gu, Jun

    2005-02-01

    Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300 mgl(-1). Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5. PMID:15717125

  18. CCHCR1 interacts specifically with the E2 protein of human papillomavirus type 16 on a surface overlapping BRD4 binding.

    PubMed

    Muller, Mandy; Demeret, Caroline

    2014-01-01

    The Human Papillomavirus E2 proteins are key regulators of the viral life cycle, whose functions are commonly mediated through protein-protein interactions with the host cell proteome. We identified an interaction between E2 and a cellular protein called CCHCR1, which proved highly specific for the HPV16 genotype, the most prevalent in HPV-associated cancers. Further characterization of the interaction revealed that CCHCR1 binds the N-terminal alpha helices of HPV16 E2 N-terminal domain. On this domain, the CCHCR1 binding interface overlaps that of BRD4, a key mediator of E2 transcriptional activity. Consequently a physical competition occurs between the two proteins for the binding to HPV16 E2, and CCHCR1 interferes with BRD4-mediated enhancement of E2-dependent transcription. In addition, we showed that the interaction with CCHCR1 induced a massive redistribution of HPV16 E2, from a predominantly nuclear to a cytoplasmic localization in dot-like structures, where E2 perfectly co-localizes with CCHCR1. Such a cytoplasmic docking likely interferes with the nuclear functions of HPV16 E2. Upon co-expression of BRD4 and CCHCR1, E2 accumulates both in the nucleus and in the cytoplasm, indicating that for HPV16, both sub-cellular localization and transcriptional functions of E2 may depend on the proportion of both factors within the cell. We provided evidence of a strong induction of the keratinocyte differentiation marker K10 by HPV16 E2, and showed that this activation is compromised by the interaction with CCHCR1. The specific interaction described here could thus impact on the pathogenesis of HPV16. We propose that it could underlie some specific traits of HPV16 infection, such as an enhanced propensity to give rise to lesions evolving toward cancer. PMID:24664238

  19. The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding

    PubMed Central

    Sharma, Garima; Upadhyay, Sandeep; Srilalitha, M.; Nandicoori, Vinay K.; Khosla, Sanjeev

    2015-01-01

    To effectively modulate the gene expression within an infected mammalian cell, the pathogen Mycobacterium tuberculosis would need to bring about epigenetic modifications at appropriate genomic loci. Working on this hypothesis, we show in this study that the mycobacterial protein Rv2966c is a 5-methylcytosine-specific DNA methyltransferase that is secreted out from the mycobacterium and gets localized to the nucleus in addition to the cytoplasm inside the host cell. Importantly, Rv2966c binds to specific DNA sequences, methylates cytosines predominantly in a non-CpG context and its methylation activity is positively influenced by phosphorylation. Interestingly, like the mammalian DNA methyltransferase, DNMT3L, Rv2966c can also interact with histone proteins. Ours is the first study that identifies a protein from a pathogenic bacteria with potential to influence host DNA methylation in a non-canonical manner providing the pathogen with a novel mechanism to alter the host epigenetic machinery. This contention is supported by repression of host genes upon M. tuberculosis infection correlated with Rv2966c binding and non-CpG methylation. PMID:25824946

  20. The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex.

    PubMed

    Brzovic, Peter S; Heikaus, Clemens C; Kisselev, Leonid; Vernon, Robert; Herbig, Eric; Pacheco, Derek; Warfield, Linda; Littlefield, Peter; Baker, David; Klevit, Rachel E; Hahn, Steven

    2011-12-23

    The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation, allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a "fuzzy" complex, where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets. PMID:22195967

  1. Transcriptional repressor E4-binding protein 4 (E4BP4) regulates metabolic hormone fibroblast growth factor 21 (FGF21) during circadian cycles and feeding.

    PubMed

    Tong, Xin; Muchnik, Marina; Chen, Zheng; Patel, Manish; Wu, Nan; Joshi, Shree; Rui, Liangyou; Lazar, Mitchell A; Yin, Lei

    2010-11-19

    Fibroblast growth factor 21 (FGF21) is a potent antidiabetic and triglyceride-lowering hormone whose hepatic expression is highly responsive to food intake. FGF21 induction in the adaptive response to fasting has been well studied, but the molecular mechanism responsible for feeding-induced repression remains unknown. In this study, we demonstrate a novel link between FGF21 and a key circadian output protein, E4BP4. Expression of Fgf21 displays a circadian rhythm, which peaks during the fasting phase and is anti-phase to E4bp4, which is elevated during feeding periods. E4BP4 strongly suppresses Fgf21 transcription by binding to a D-box element in the distal promoter region. Depletion of E4BP4 in synchronized Hepa1c1c-7 liver cells augments the amplitude of Fgf21 expression, and overexpression of E4BP4 represses FGF21 secretion from primary mouse hepatocytes. Mimicking feeding effects, insulin significantly increases E4BP4 expression and binding to the Fgf21 promoter through AKT activation. Thus, E4BP4 is a novel insulin-responsive repressor of FGF21 expression during circadian cycles and feeding. PMID:20851878

  2. Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography.

    PubMed

    Bian, Liujiao; Li, Qian; Ji, Xu

    2015-07-01

    The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands. PMID:25981289

  3. The linker connecting the two kringles plays a key role in prothrombin activation

    PubMed Central

    Pozzi, Nicola; Chen, Zhiwei; Pelc, Leslie A.; Shropshire, Daniel B.; Di Cera, Enrico

    2014-01-01

    The zymogen prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction that is accelerated >3,000-fold by cofactor Va. This physiologically important effect is paradigmatic of analogous cofactor-dependent reactions in the coagulation and complement cascades, but its structural determinants remain poorly understood. Prothrombin has three linkers connecting the N-terminal Gla domain to kringle-1 (Lnk1), the two kringles (Lnk2), and kringle-2 to the C-terminal protease domain (Lnk3). Recent developments indicate that the linkers, and particularly Lnk2, confer on the zymogen significant flexibility in solution and enable prothrombin to sample alternative conformations. The role of this flexibility in the context of prothrombin activation was tested with several deletions. Removal of Lnk2 in almost its entirety (ProTΔ146–167) drastically reduces the enhancement of thrombin generation by cofactor Va from >3,000-fold to 60-fold because of a significant increase in the rate of activation in the absence of cofactor. Deletion of Lnk2 mimics the action of cofactor Va and offers insights into how prothrombin is activated at the molecular level. The crystal structure of ProTΔ146–167 reveals a contorted architecture where the domains are not vertically stacked, kringle-1 comes within 9 Å of the protease domain, and the Gla-domain primed for membrane binding comes in contact with kringle-2. These findings broaden our molecular understanding of a key reaction of the blood coagulation cascade where cofactor Va enhances activation of prothrombin by factor Xa by compressing Lnk2 and morphing prothrombin into a conformation similar to the structure of ProTΔ146–167. PMID:24821807

  4. Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting-out.

    PubMed

    Bian, Liujiao; Ji, Xu; Hu, Wei

    2014-07-01

    In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine-tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni-chelated Sepharose Fast-Flow affinity column, ammonium sulfate salting-out and Sephadex G-75 size-exclusion column in turn. The purity analysis by SDS-PAGE, high-performance size-exclusion and reversed-phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti-angiogenic activity under the effective range of 5.0-25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%. PMID:24311387

  5. Structure–activity relationships of the human prothrombin kringle-2 peptide derivative NSA9: anti-proliferative activity and cellular internalization

    PubMed Central

    Hwang, Hyun Sook; Kim, Dong Won; Kim, Soung Soo

    2006-01-01

    The human prothrombin kringle-2 protein inhibits angiogenesis and LLC (Lewis lung carcinoma) growth and metastasis in mice. Additionally, the NSA9 peptide (NSAVQLVEN) derived from human prothrombin kringle-2 has been reported to inhibit the proliferation of BCE (bovine capillary endothelial) cells and CAM (chorioallantoic membrane) angiogenesis. In the present study, we examined the structure–activity relationships of the NSA9 peptide in inhibiting the proliferation of endothelial cells lines e.g. BCE and HUVE (human umbilical vein endothelial). N- or C-terminal truncated derivatives and reverse sequence analogues of NSA9 were prepared and their anti-proliferative activities were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. This cell proliferation assay demonstrated that both the N-terminal region and sequence orientation of NSA9 are important for inhibiting the proliferation of endothelial cells. In particular 2 C-terminal truncation derivatives of NSA9 [NSA7 (NSAVQLV) and NSA8 (NSAVQLVE)] inhibited cellular proliferation to a greater extent than did NSA9. The heptapeptide NSA7, was found to be more potent than NSA9 in inhibiting CAM angiogenesis, and tubular formation and migration of HUVE cells. In addition NSA9, NSA8 and NSA7 peptides exhibited considerable inhibitory effects on the proliferation of tumour cells such as B16F10 (murine melanoma), LLC and L929 (murine fibroblast). Also, cellular internalization studies demonstrated that NSA7 was internalized into both endothelial and tumour cells more easily than was NSA9. In conclusion, these results suggest that NSA7, residing within the full sequence of NSA9, contains the required sequence for anti-proliferative activity and cellular internalization. PMID:16390327

  6. Prothrombin kringle-2 induces death of mesencephalic dopaminergic neurons in vivo and in vitro via microglial activation.

    PubMed

    Kim, Sang Ryong; Chung, Eun Sook; Bok, Eugene; Baik, Hyung Hwan; Chung, Young Cheul; Won, So Yoon; Joe, Eunhye; Kim, Tae Hyong; Kim, Soung Soo; Jin, Min Young; Choi, Sang Ho; Jin, Byung Kwan

    2010-05-15

    We have shown that prothrombin kringle-2 (pKr-2), a domain of human prothrombin distinct from thrombin could activate cultured rat brain microglia in vitro. However, little is known whether pKr-2-induced microglial activation could cause neurotoxicity on dopaminergic (DA) neurons in vivo. To address this question, pKr-2 was injected into the rat substantia nigra (SN). Tyrosine hydroxylase (TH) immunohistochemistry experiments demonstrate significant loss of DA neurons seven days after injection of pKr-2. In parallel, pKr-2-activated microglia were detected in the SN with OX-42 and OX-6 immunohistochemistry. Reverse transcription PCR and double-label immunohistochemistry revealed that activated microglia in vivo exhibit early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and several proinflammatory cytokines. The pKr-2-induced loss of SN DA neurons was partially inhibited by the NOS inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride, and the COX-2 inhibitor DuP-697. Extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were activated in the SN as early as 1 hr after pKr-2 injection, and localized within microglia. Inhibition of these kinases led to attenuation of mRNA expression of iNOS, COX-2 and several proinflammatory cytokines, and rescue of DA neurons in the SN. Intriguingly, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of mesencephalic neurons and microglia, but not microglia-free neuron-enriched mesencephalic cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that microglia activated by endogenous compound(s), such as pKr-2, are implicated in the DA neuronal cell death in the SN. PMID:20025058

  7. Prothrombin Kringle-2: A Potential Inflammatory Pathogen in the Parkinsonian Dopaminergic System

    PubMed Central

    Leem, Eunju; Jeong, Kyoung Hoon; Won, So-Yoon

    2016-01-01

    Although accumulating evidence suggests that microglia-mediated neuroinflammation may be crucial for the initiation and progression of Parkinson's disease (PD), and that the control of neuroinflammation may be a useful strategy for preventing the degeneration of nigrostriatal dopaminergic (DA) projections in the adult brain, it is still unclear what kinds of endogenous biomolecules initiate microglial activation, consequently resulting in neurodegeneration. Recently, we reported that the increase in the levels of prothrombin kringle-2 (pKr-2), which is a domain of prothrombin that is generated by active thrombin, can lead to disruption of the nigrostriatal DA projection. This disruption is mediated by neurotoxic inflammatory events via the induction of microglial Toll-like receptor 4 (TLR4) in vivo , thereby resulting in less neurotoxicity in TLR4-deficient mice. Moreover, inhibition of microglial activation following minocycline treatment, which has anti-inflammatory activity, protects DA neurons from pKr-2-induced neurotoxicity in the substantia nigra (SN) in vivo. We also found that the levels of pKr-2 and microglial TLR4 were significantly increased in the SN of PD patients compared to those of age-matched controls. These observations suggest that there may be a correlation between pKr-2 and microglial TLR4 in the initiation and progression of PD, and that inhibition of pKr-2-induced microglial activation may be protective against the degeneration of the nigrostriatal DA system in vivo. To describe the significance of pKr-2 overexpression, which may have a role in the pathogenesis of PD, we have reviewed the mechanisms of pKr-2-induced microglial activation, which results in neurodegeneration in the SN of the adult brain. PMID:27574481

  8. Prothrombin Kringle-2: A Potential Inflammatory Pathogen in the Parkinsonian Dopaminergic System.

    PubMed

    Leem, Eunju; Jeong, Kyoung Hoon; Won, So-Yoon; Shin, Won-Ho; Kim, Sang Ryong

    2016-08-01

    Although accumulating evidence suggests that microglia-mediated neuroinflammation may be crucial for the initiation and progression of Parkinson's disease (PD), and that the control of neuroinflammation may be a useful strategy for preventing the degeneration of nigrostriatal dopaminergic (DA) projections in the adult brain, it is still unclear what kinds of endogenous biomolecules initiate microglial activation, consequently resulting in neurodegeneration. Recently, we reported that the increase in the levels of prothrombin kringle-2 (pKr-2), which is a domain of prothrombin that is generated by active thrombin, can lead to disruption of the nigrostriatal DA projection. This disruption is mediated by neurotoxic inflammatory events via the induction of microglial Toll-like receptor 4 (TLR4) in vivo , thereby resulting in less neurotoxicity in TLR4-deficient mice. Moreover, inhibition of microglial activation following minocycline treatment, which has anti-inflammatory activity, protects DA neurons from pKr-2-induced neurotoxicity in the substantia nigra (SN) in vivo. We also found that the levels of pKr-2 and microglial TLR4 were significantly increased in the SN of PD patients compared to those of age-matched controls. These observations suggest that there may be a correlation between pKr-2 and microglial TLR4 in the initiation and progression of PD, and that inhibition of pKr-2-induced microglial activation may be protective against the degeneration of the nigrostriatal DA system in vivo. To describe the significance of pKr-2 overexpression, which may have a role in the pathogenesis of PD, we have reviewed the mechanisms of pKr-2-induced microglial activation, which results in neurodegeneration in the SN of the adult brain. PMID:27574481

  9. Apolipoprotein(a) Kringle-IV Type 2 Copy Number Variation Is Associated with Venous Thromboembolism

    PubMed Central

    Sticchi, Elena; Magi, Alberto; Kamstrup, Pia R.; Marcucci, Rossella; Prisco, Domenico; Martinelli, Ida; Mannucci, Pier Mannuccio; Abbate, Rosanna; Giusti, Betti

    2016-01-01

    In addition to the established association between high lipoprotein(a) [Lp(a)] concentrations and coronary artery disease, an association between Lp(a) and venous thromboembolism (VTE) has also been described. Lp(a) is controlled by genetic variants in LPA gene, coding for apolipoprotein(a), including the kringle-IV type 2 (KIV-2) size polymorphism. Aim of the study was to investigate the role of LPA gene KIV-2 size polymorphism and single nucleotide polymorphisms (SNPs) (rs1853021, rs1800769, rs3798220, rs10455872) in modulating VTE susceptibility. Five hundred and sixteen patients with VTE without hereditary and acquired thrombophilia and 1117 healthy control subjects, comparable for age and sex, were investigated. LPA KIV-2 polymorphism, rs3798220 and rs10455872 SNPs were genotyped by TaqMan technology. Concerning rs1853021 and rs1800769 SNPs, PCR-RFLP assay was used. LPA KIV-2 repeat number was significantly lower in patients than in controls [median (interquartile range) 11(6–17) vs 15(9–25), p<0.0001]. A significantly higher prevalence of KIV-2 repeat number ≤7 was observed in patients than in controls (33.5% vs 15.5%, p<0.0001). KIV-2 repeat number was independently associated with VTE (p = 4.36 x10-9), as evidenced by the general linear model analysis adjusted for transient risk factors. No significant difference in allele frequency for all SNPs investigated was observed. Haplotype analysis showed that LPA haplotypes rather than individual SNPs influenced disease susceptibility. Receiver operating characteristic curves analysis showed that a combined risk prediction model, including KIV-2 size polymorphism and clinical variables, had a higher performance in identifying subjects at VTE risk than a clinical-only model, also separately in men and women. PMID:26900838

  10. Apolipoprotein(a) Kringle-IV Type 2 Copy Number Variation Is Associated with Venous Thromboembolism.

    PubMed

    Sticchi, Elena; Magi, Alberto; Kamstrup, Pia R; Marcucci, Rossella; Prisco, Domenico; Martinelli, Ida; Mannucci, Pier Mannuccio; Abbate, Rosanna; Giusti, Betti

    2016-01-01

    In addition to the established association between high lipoprotein(a) [Lp(a)] concentrations and coronary artery disease, an association between Lp(a) and venous thromboembolism (VTE) has also been described. Lp(a) is controlled by genetic variants in LPA gene, coding for apolipoprotein(a), including the kringle-IV type 2 (KIV-2) size polymorphism. Aim of the study was to investigate the role of LPA gene KIV-2 size polymorphism and single nucleotide polymorphisms (SNPs) (rs1853021, rs1800769, rs3798220, rs10455872) in modulating VTE susceptibility. Five hundred and sixteen patients with VTE without hereditary and acquired thrombophilia and 1117 healthy control subjects, comparable for age and sex, were investigated. LPA KIV-2 polymorphism, rs3798220 and rs10455872 SNPs were genotyped by TaqMan technology. Concerning rs1853021 and rs1800769 SNPs, PCR-RFLP assay was used. LPA KIV-2 repeat number was significantly lower in patients than in controls [median (interquartile range) 11(6-17) vs 15(9-25), p<0.0001]. A significantly higher prevalence of KIV-2 repeat number ≤7 was observed in patients than in controls (33.5% vs 15.5%, p<0.0001). KIV-2 repeat number was independently associated with VTE (p = 4.36 x10-9), as evidenced by the general linear model analysis adjusted for transient risk factors. No significant difference in allele frequency for all SNPs investigated was observed. Haplotype analysis showed that LPA haplotypes rather than individual SNPs influenced disease susceptibility. Receiver operating characteristic curves analysis showed that a combined risk prediction model, including KIV-2 size polymorphism and clinical variables, had a higher performance in identifying subjects at VTE risk than a clinical-only model, also separately in men and women. PMID:26900838

  11. Anti-angiogenic action of plasma hyaluronan binding protein in human umbilical vein endothelial cells.

    PubMed

    Jeon, Ji Won; Song, Hyun Seok; Moon, Eun-Joung; Park, Shi-Young; Son, Myung Jin; Jung, Seung Youn; Kim, Ji Tae; Nam, Do-Hyun; Choi-Miura, Nam-Ho; Kim, Kyu-Won; Kim, Yung-Jin

    2006-07-01

    The kringle domain is a triple loop structure present in angiostatin and endostatin. The disulfide bond-linked kringle architectures have been known to be essential for anti-angiogenic activity. Plasma hyaluronan binding protein (PHBP) is a novel serine protease which consists of three epidermal growth factor (EGF) domains, a kringle domain, and a serine protease domain. PHBP can be cleaved autocatalytically to generate activity and is highly expressed in the human blood and liver. To determine the anti-angiogenic activities of PHBP, we purified recombinant mouse PHBP from stable cell line overexpressing PHBP and used protein in vivo and in vitro angiogenesis assays. We found that recombinant PHBP inhibits not only angiogenesis in vivo in chorioallantoic membrane (CAM) assay but also the basic fibroblast growth factor (bFGF)-induced proliferation, invasion and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependant manner. Moreover, we found that the kringle domain of PHBP was essential for the anti-angiogenic action of PHBP by the deletion mutants. These findings unravel a new function of PHBP as an inhibitor of the proangiogenic phenotype of vascular endothelial cells and demonstrate that the kringle domain of PHBP might be a potent novel inhibitor of activated endothelial cells in vitro and in vivo. PMID:16773202

  12. Suppression of Colorectal Cancer Liver Metastasis by Apolipoprotein(a) Kringle V in a Nude Mouse Model through the Induction of Apoptosis in Tumor-Associated Endothelial Cells

    PubMed Central

    Ahn, Jin-Hyung; Yu, Hyun-Kyung; Lee, Ho-Jeong; Hong, Soon Won; Kim, Sun Jin; Kim, Jang-Seong

    2014-01-01

    The formation of liver metastases in colorectal cancer patients is the primary cause of patient death. Current therapies directed at liver metastasis from colorectal cancer have had minimal impact on patient outcomes. Therefore, the development of alternative treatment strategies for liver metastasis is needed. In the present study, we demonstrated that recombinant human apolipoprotein(a) kringle V, also known as rhLK8, induced the apoptotic turnover of endothelial cells in vitro through the mitochondrial apoptosis pathway. The interaction of rhLK8 with glucose-regulated protein 78 (GRP78) may be involved in the induction of apoptosis because the inhibition of GRP78 by GRP78-specific antibodies or siRNA knockdown inhibited the rhLK8-mediated apoptosis of human umbilical vein endothelial cells in vitro. Next, to evaluate the effects of rhLK8 on angiogenesis and metastasis, an experimental model of liver metastasis was established by injecting a human colorectal cancer cell line, LS174T, into the spleens of BALB/c nude mice. The systemic administration of rhLK8 significantly suppressed liver metastasis from human colorectal cancer cells and improved host survival compared with controls. The combination of rhLK8 and 5-fluorouracil substantially increased these survival benefits compared with either therapy alone. Histological observation showed significant induction of apoptosis among tumor-associated endothelial cells in liver metastases from rhLK8-treated mice compared with control mice. Collectively, these results suggest that the combination of rhLK8 with conventional chemotherapy may be a promising approach for the treatment of patients with life-threatening colorectal cancer liver metastases. PMID:24699568

  13. The major cockroach allergen Bla g 4 binds tyramine and octopamine.

    PubMed

    Offermann, Lesa R; Chan, Siew Leong; Osinski, Tomasz; Tan, Yih Wan; Chew, Fook Tim; Sivaraman, J; Mok, Yu-Keung; Minor, Wladek; Chruszcz, Maksymilian

    2014-07-01

    Bla g 4 is a male cockroach specific protein and is one of the major allergens produced by Blattella germanica (German cockroach). This protein belongs to the lipocalin family that comprises a set of proteins that characteristically bind small hydrophobic molecules and play a role in a number of processes such as: retinoid and pheromone transport, prostaglandin synthesis and mammalian immune response. Using NMR and isothermal titration calorimetry we demonstrated that Bla g 4 binds tyramine and octopamine in solution. In addition, crystal structure analysis of the complex revealed details of tyramine binding. As tyramine and octopamine play important roles in invertebrates, and are counterparts to vertebrate adrenergic transmitters, we speculate that these molecules are physiological ligands for Bla g 4. The nature of binding these ligands to Bla g 4 sheds light on the possible biological function of the protein. In addition, we performed a large-scale analysis of Bla g 4 and Per a 4 (an allergen from American cockroach) homologs to get insights into the function of these proteins. This analysis together with a structural comparison of Blag 4 and Per a 4 suggests that these proteins may play different roles and most likely bind different ligands. Accession numbers: The atomic coordinates and the structure factors have been deposited to the Protein Data Band under accession codes: 4N7C for native Bla g 4 and 4N7D for the Se-Met Bla g 4 structure. PMID:24769496

  14. CD4 Binding Affinity Determines Human Immunodeficiency Virus Type 1-Induced Alpha Interferon Production in Plasmacytoid Dendritic Cells ▿

    PubMed Central

    Haupt, Sabrina; Donhauser, Norbert; Chaipan, Chawaree; Schuster, Philipp; Puffer, Bridget; Daniels, Rod S.; Greenough, Thomas C.; Kirchhoff, Frank; Schmidt, Barbara

    2008-01-01

    Plasmacytoid dendritic cells (PDC) are major producers of type I interferons (IFN) in response to human immunodeficiency virus type 1 (HIV-1) infection. To better define the underlying mechanisms, we studied the magnitude of alpha IFN (IFN-α) induction by recombinant viruses containing changes in the Env protein that impair or disrupt CD4 binding or expressing primary env alleles with differential coreceptor tropism. We found that the CD4 binding affinity but not the viral coreceptor usage is critical for the attachment of autofluorescing HIV-1 to PDC and for subsequent IFN-α induction. Our results illustrate the importance of the gp120-CD4 interaction in determining HIV-1-induced immune stimulation via IFN-α production. PMID:18579609

  15. Induction of microglial toll-like receptor 4 by prothrombin kringle-2: a potential pathogenic mechanism in Parkinson's disease.

    PubMed

    Shin, Won-Ho; Jeon, Min-Tae; Leem, Eunju; Won, So-Yoon; Jeong, Kyoung Hoon; Park, Sang-Joon; McLean, Catriona; Lee, Sung Joong; Jin, Byung Kwan; Jung, Un Ju; Kim, Sang Ryoung

    2015-01-01

    Microglia-mediated neuroinflammation may play an important role in the initiation and progression of dopaminergic (DA) neurodegeneration in Parkinson's disease (PD), and toll-like receptor 4 (TLR4) is essential for the activation of microglia in the adult brain. However, it is still unclear whether patients with PD exhibit an increase in TLR4 expression in the brain, and whether there is a correlation between the levels of prothrombin kringle-2 (pKr-2) and microglial TLR4. In the present study, we first observed that the levels of pKr-2 and microglial TLR4 were increased in the substantia nigra (SN) of patients with PD. In rat and mouse brains, intranigral injection of pKr-2, which is not directly toxic to neurons, led to the disruption of nigrostriatal DA projections. Moreover, microglial TLR4 was upregulated in the rat SN and in cultures of the BV-2 microglial cell line after pKr-2 treatment. In TLR4-deficient mice, pKr-2-induced microglial activation was suppressed compared with wild-type mice, resulting in attenuated neurotoxicity. Therefore, our results suggest that pKr-2 may be a pathogenic factor in PD, and that the inhibition of pKr-2-induced microglial TLR4 may be protective against degeneration of the nigrostriatal DA system in vivo. PMID:26440368

  16. Inhibition of prothrombin kringle-2-induced inflammation by minocycline protects dopaminergic neurons in the substantia nigra in vivo.

    PubMed

    Nam, Jin Han; Leem, Eunju; Jeon, Min-Tae; Kim, Young-Je; Jung, Un Ju; Choi, Myung-Sook; Maeng, Sungho; Jin, Byung Kwan; Kim, Sang Ryong

    2014-05-01

    Prothrombin kringle-2 (pKr-2), a domain of prothrombin, can cause the degeneration of mesencephalic dopaminergic neurons through microglial activation. However, the chemical products that inhibit pKr-2-induced inflammatory activities in the brain are still not well known. The present study investigated whether minocycline, a semisynthetic tetracycline derivative, could inhibit pKr-2-induced microglial activation and prevent the loss of nigral dopaminergic (DA) neurons in vivo. To address this question, rats were administered a unilateral injection of pKr-2 in the substantia nigra in the presence or absence of minocycline. Our results show that pKr-2 induces the production of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and inducible nitric oxide synthase from the activated microglia. In parallel, 7 days after pKr-2 injection, tyrosine hydroxylase immunocytochemical analysis and western blot analysis showed a significant loss of nigral DA neurons. This neurotoxicity was antagonized by minocycline and the observed neuroprotective effects were associated with the ability of minocycline to suppress the expression of tumor necrosis factor-α, interleukin-1β, and nitric oxide synthase. These results suggest that minocycline may be promising as a potential therapeutic agent for the prevention of DA neuronal degeneration associated with pKr-2-induced microglial activation. PMID:24488033

  17. Ablation of nectin4 binding compromises CD46 usage by a hybrid vesicular stomatitis virus/measles virus.

    PubMed

    Liu, Yu-Ping; Russell, Samuel P; Ayala-Breton, Camilo; Russell, Stephen J; Peng, Kah-Whye

    2014-02-01

    Measles virus (MV) immunosuppression is due to infection of SLAM-positive immune cells, whereas respiratory shedding and virus transmission are due to infection of nectin4-positive airway epithelial cells. The vaccine lineage MV strain Edmonston (MV-Edm) acquired an additional tropism for CD46 which is the basis of its oncolytic specificity. VSVFH is a vesicular stomatitis virus (VSV) encoding the MV-Edm F and H entry proteins in place of G. The virus spreads faster than MV-Edm and is highly fusogenic and a potent oncolytic. To determine whether ablating nectin4 tropism from VSVFH might prevent shedding, increasing its safety profile as an oncolytic, or might have any effect on CD46 binding, we generated VSVFH viruses with H mutations that disrupt attachment to SLAM and/or nectin4. Disruption of nectin4 binding reduced release of VSVFH from the basolateral side of differentiated airway epithelia composed of Calu-3 cells. However, because nectin4 and CD46 have substantially overlapping receptor binding surfaces on H, disruption of nectin4 binding compromised CD46 binding and greatly diminished the oncolytic potency of these viruses on human cancer cells. Thus, our results support continued preclinical development of VSVFH without ablation of nectin4 binding. PMID:24335299

  18. Hierarchical Oct4 Binding in Concert with Primed Epigenetic Rearrangements during Somatic Cell Reprogramming.

    PubMed

    Chen, Jun; Chen, Xiaolong; Li, Min; Liu, Xiaoyu; Gao, Yawei; Kou, Xiaochen; Zhao, Yanhong; Zheng, Weisheng; Zhang, Xiaobai; Huo, Yi; Chen, Chuan; Wu, You; Wang, Hong; Jiang, Cizhong; Gao, Shaorong

    2016-02-16

    The core pluripotency factor Oct4 plays key roles in somatic cell reprogramming through transcriptional control. Here, we profile Oct4 occupancy, epigenetic changes, and gene expression in reprogramming. We find that Oct4 binds in a hierarchical manner to target sites with primed epigenetic modifications. Oct4 binding is temporally continuous and seldom switches between bound and unbound. Oct4 occupancy in most of promoters is maintained throughout the entire reprogramming process. In contrast, somatic cell-specific enhancers are silenced in the early and intermediate stages, whereas stem cell-specific enhancers are activated in the late stage in parallel with cell fate transition. Both epigenetic remodeling and Oct4 binding contribute to the hyperdynamic enhancer signature transitions. The hierarchical Oct4 bindings are associated with distinct functional themes at different stages. Collectively, our results provide a comprehensive molecular roadmap of Oct4 binding in concert with epigenetic rearrangements and rich resources for future reprogramming studies. PMID:26832419

  19. Antiangiogenic Therapy with Human Apolipoprotein(a) Kringle V and Paclitaxel in a Human Ovarian Cancer Mouse Model12

    PubMed Central

    Yu, Hyun-Kyung; Lee, Ho-Jeong; Yun, Seok-Joong; Lee, Sun-Joo; Langley, Robert R.; Yoon, Yeup; Yi, Lee S.H.; Bae, Duk-Soo; Kim, Jang-Seong; Kim, Sun Jin

    2014-01-01

    INTRODUCTION: The present study compared the effect of combination therapy using human apolipoprotein(a) kringle V (rhLK8) to conventional chemotherapy with paclitaxel for human ovarian carcinoma producing high or low levels of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: Human ovarian carcinoma cells producing high (SKOV3ip1) or low (HeyA8) levels of VEGF were implanted into the peritoneal cavity of female nude mice. Seven days later, mice were randomized into four groups: control (vehicle), paclitaxel [5 mg/kg, weekly intraperitoneal (i.p.) injection], rhLK8 (50 mg/kg, daily i.p. injection), or the combination of paclitaxel and rhLK8. Mice were treated for 4 weeks and examined by necropsy. RESULTS: In mice implanted with SKOV3ip1 cells, rhLK8 treatment had no significant effect on tumor incidence or the volume of ascites but induced a significant decrease in tumor weight compared with control mice. Paclitaxel significantly reduced tumor weight and ascites volume, and combination treatment with paclitaxel and rhLK8 had an additive therapeutic effect. Similarly, in HeyA8 mice, the effect of combination treatment on tumor weight and tumor incidence was statistically significantly greater than that of paclitaxel or rhLK8 alone. Immunohistochemical analysis showed a significant decrease in microvessel density and a marked increase of apoptosis in tumor and tumor-associated endothelial cells in response to combination treatment with paclitaxel and rhLK8. CONCLUSION: Collectively, these results suggest that antiangiogenic therapy with rhLK8 in combination with taxane-based conventional chemotherapy could be effective for the treatment of ovarian carcinomas, regardless of VEGF status. PMID:25180060

  20. Conformation of lys-plasminogen and the Kringle 1-3 fragment of plasminogen analyzed by small-angle neutron scattering

    SciTech Connect

    Ramakrishnan, V.; Mangel, W.F. ); Patthy, L. )

    1991-04-23

    Native human Glu-plasminogen (Glu{sub 1}-Asn{sub 791}) was previously shown to have a radius of gyration of 39{angstrom} and a shape best described by a prolate ellipsoid. Upon occupation of a weak lysine-binding site, the shape reversibly changes to that best described by a Debye random coil with a radius of gyration of 56{angstrom}. Conversion from the closed to the open form is not accompanied by any change in secondary structure, hence the closed conformation is formed by interaction between domains, the five kringles and the protease domain, and this is abolished upon conversion to the open form. Here the authors analyzed by small-angle neutron scattering the conformations of human Lys-plasminogen (Lys{sub 78}-Asn{sub 791}) and the fragment K1-3 that contains the first three kringles of plasminogen (Tyr{sub 80}-Val{sub 338} or Tyr{sub 80}-Val{sub 354}). The fragment K1-3 in the absence or presence of 6-aminohexanoic acid had a shape best described equally either by an elongated prolate ellipsoid or by a Debye random coil, with a radius of gyration of 29{angstrom}. They model for the two forms of plasminogen is that, in the closed form, domain interaction generates a compact, almost globular, structure. The structural differences between the closed and open forms of plasminogen are discussed in terms of functional differences relevant to the regulation of plasminogen activation.

  1. Competition for in vitro ( sup 3 H)gibberellin A sub 4 binding in cucumber by substituted phthalimides

    SciTech Connect

    Yalpani, N.; Suttle, J.C.; Hultstrand, J.F. ); Rodaway, S.J. )

    1989-11-01

    Certain N-substituted phthalimides (NSPs) have gibberellin (GA)-like activity in a number of GA bioassays. The interaction between representative NSPs and a protein fraction from cucumber (Cucumis sativus L.) hypocotyls that has GA-binding characteristics consistent with those expected of GA receptors was studied. Analysis of in vitro equilibrium saturation data indicated the presence of only one class of high affinity ({sup 3}H)GA{sub 4} binding sites (K{sub d} {approximately}30 nanomolar, n = 0.25 picomole per milligram of protein). In the presence of 6 or 60 micromolar 1-(3-chlorophthalimido)-cyclohexanecarboximide (AC-94,377), the K{sub d} for ({sup 3}H)GA{sub 4} increased, whereas the maximum number of saturable ({sup 3}H)GA{sub 4} binding sites did not change significantly. The dissociation of ({sup 3}H)GA{sub 4} from its binding sites was complex and was best described by a bi-exponential equation. AC-94,377 did not affect the rates of ({sup 3}H)GA{sub 4} dissociation from its binding sites. These results implied that AC-94,377 and ({sup 3}H)GA{sub 4} compete for binding to the same sites. A correlation was observed between the activity of over 20 NSPs in the cucumber hypocotyl bioassay and their in vitro affinity for the GA binding sites. Our observations lend further support to the notion that certain GA binding proteins in cucumber cytosol are GA receptors and also provide a molecular explanation for the GA-like in vivo activity of some NSPs.

  2. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    SciTech Connect

    Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

    2015-02-15

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.

  3. TRPA1 Is Functionally Expressed Primarily by IB4-Binding, Non-Peptidergic Mouse and Rat Sensory Neurons

    PubMed Central

    Stucky, Cheryl L.

    2012-01-01

    Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter (<27 µm) neurons from lumbar 1–6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2–4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter) responded to AITC (6%) or cinnamaldehyde (4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is functionally

  4. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    PubMed Central

    Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

    2014-01-01

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. PMID:25555152

  5. Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022.

    PubMed

    Kohnert, U; Rudolph, R; Verheijen, J H; Weening-Verhoeff, E J; Stern, A; Opitz, U; Martin, U; Lill, H; Prinz, H; Lechner, M

    1992-01-01

    BM 06.022 is a t-PA deletion variant which comprises the kringle 2 and the protease domain. Production of BM 06.022 in Escherichia coli leads to the formation of inactive inclusion bodies, which have to be refolded by an in vitro refolding process to achieve activity and proper structure of the domains. We analysed the biochemical properties of BM 06.022 to obtain some information about the structure of kringle 2 and the protease as compared with the structure of these domains in the intact t-PA molecule. The kinetic analysis of the amidolytic activity of BM 06.022 and CHO-t-PA yielded similar values for kcat (13.9 s-1 and 11.4 s-1 for the single chain forms and 33.9 s-1 and 27.1 s-1 for the two chain forms of BM 06.022 and CHO-t-PA, respectively) and for Km (2.5 mM and 2.1 mM for the single chains forms and 0.5 mM and 0.3 mM for the two chain forms of BM 06.022 and CHO-t-PA, respectively). BM 06.022 and CHO-t-PA have the same plasminogenolytic activity in the absence of CNBr fragments of fibrinogen. However, BM 06.022 has a lower plasminogenolytic activity in the presence of CNBr fragments of fibrinogen and a lower affinity to fibrin as compared with CHO-t-PA. The affinity of BM 06.022 for fibrin is completely suppressed by 0.3 mM epsilon-aminocaproic acid, while the intact t-PA has a residual affinity of approximately 30%. The dissociation constants for the interaction with the lysine analogue epsilon-aminocaproic acid are 0.10 mM and 0.09 mM for BM 06.022 and the intact t-PA, respectively. Furthermore, BM 06.022 and CHO-t-PA are inhibited by PAI-1 in a similar manner. PMID:1321420

  6. Mast cell degranulation is negatively regulated by the Munc13-4-binding small-guanosine triphosphatase Rab37

    PubMed Central

    Higashio, Hironori; Satoh, Yoh-ichi; Saino, Tomoyuki

    2016-01-01

    Mast cell degranulation is regulated by the small guanosine triphosphatases (GTPases) Rab27a and Rab27b, which have distinct and opposing roles: Rab27b acts as a positive regulator through its effector protein Munc13-4, a non-neuronal isoform of the vesicle-priming Munc13 family of proteins, whereas Rab27a acts as a negative regulator through its effector protein melanophilin, by maintaining integrity of cortical filamentous actin (F-actin), a barrier to degranulation. Here we investigated the role of Rab37, one of the Rab GTPases assumed to be implicated in regulated secretion during mast cell degranulation. Using the RBL-2H3 mast cell line, we detected Rab37 on the secretory granules and found that antigen-induced degranulation was extensively increased by either knockdown of Rab37 or overexpression of a dominant-active Rab37 mutant. This hypersecretion phenotype in the Rab37-knockdown cells was suppressed by simultaneous knockdown of Rab27a and Rab27b or of Munc13-4, but not by disruption of cortical F-actin. We further found that Rab37 interacted with Munc13-4 in a GTP-independent manner and formed a Rab27-Munc13-4-Rab37 complex. These results suggest that Rab37 is a Munc13-4-binding protein that inhibits mast cell degranulation through its effector protein, by counteracting the vesicle-priming activity of the Rab27-Munc13-4 system. PMID:26931073

  7. The human prothrombin kringle-2 derived peptide, NSA9, is internalized into bovine capillary endothelial cells through endocytosis and energy-dependent pathways

    SciTech Connect

    Hwang, Hyun Sook; Kim, Soung Soo . E-mail: kimss518@yonsei.ac.kr

    2005-09-23

    Human prothrombin kringle-2 and its partial peptide, NSA9 (NSAVQLVEN), have been reported to have potent anti-angiogenic activities. Here, the internalization mechanism of NSA9 into bovine capillary endothelial (BCE) cells was examined using lactate dehydrogenase (LDH) release assay, fluorescence microscopy, and flow cytometry. LDH release assay results suggested that the integrity of the BCE cell membrane was unaffected by NSA9. Fluorescence microscopy indicated that internalized NSA9 was localized in the cytoplasm around the nucleus, and showed a punctuated fluorescence pattern, which is indicative of endocytic vesicles. Also, the cellular internalization of NSA9 is significantly inhibited by depletion of the cellular ATP pool, endocytosis inhibitors such as chloroquine and nocodazole, and incubation at low temperature (4 deg C). In addition, the anti-proliferative activity of NSA9 against BCE cells was diminished in the presence of endocytosis or metabolic inhibitors. In conclusion, these results strongly suggest that NSA9 might exert its anti-proliferative activity through internalization into BCE cells by endocytosis and energy-dependent pathways.

  8. In vitro and in vivo antiangiogenic activity of a novel deca-peptide derived from human tissue-type plasminogen activator kringle 2

    SciTech Connect

    Su, Li; Xu, Xun; Zhao, Hui; Gu, Qing; Zou, Haidong

    2010-06-11

    A synthetic deca-peptide corresponding to the amino acid sequence Arg{sup 54}-Trp{sup 63} of human tissue-type plasminogen activator (t-PA) kringle 2 domain, named TKII-10, is produced and tested for its ability to inhibit endothelial cell proliferation, migration, tube formation in vitro, and angiogenesis in vivo. At the same time, another peptide TKII-10S composed of the same 10 amino acids as TKII-10, but in a different sequence, is also produced and tested. The results show that TKII-10 potently inhibits VEGF-stimulated endothelial cell migration and tube formation in a dose-dependent, as well as sequence-dependent, manner in vitro while it is inactive in inhibiting endothelial cell proliferation. Furthermore, TKII-10 potently inhibits angiogenesis in chick chorioallantoic membrane and mouse cornea. The middle four amino acids DGDA in their sequence play an important role in TKII-10 angiogenesis inhibition{sub .} These results suggest that TKII-10 is a novel angiogenesis inhibitor that may serve as a prototype for antiangiogenic drug development.

  9. Induction of microglial toll-like receptor 4 by prothrombin kringle-2: a potential pathogenic mechanism in Parkinson’s disease

    PubMed Central

    Shin, Won-Ho; Jeon, Min-Tae; Leem, Eunju; Won, So-Yoon; Jeong, Kyoung Hoon; Park, Sang-Joon; McLean, Catriona; Lee, Sung Joong; Jin, Byung Kwan; Jung, Un Ju; Kim, Sang Ryoung

    2015-01-01

    Microglia-mediated neuroinflammation may play an important role in the initiation and progression of dopaminergic (DA) neurodegeneration in Parkinson’s disease (PD), and toll-like receptor 4 (TLR4) is essential for the activation of microglia in the adult brain. However, it is still unclear whether patients with PD exhibit an increase in TLR4 expression in the brain, and whether there is a correlation between the levels of prothrombin kringle-2 (pKr-2) and microglial TLR4. In the present study, we first observed that the levels of pKr-2 and microglial TLR4 were increased in the substantia nigra (SN) of patients with PD. In rat and mouse brains, intranigral injection of pKr-2, which is not directly toxic to neurons, led to the disruption of nigrostriatal DA projections. Moreover, microglial TLR4 was upregulated in the rat SN and in cultures of the BV-2 microglial cell line after pKr-2 treatment. In TLR4-deficient mice, pKr-2-induced microglial activation was suppressed compared with wild-type mice, resulting in attenuated neurotoxicity. Therefore, our results suggest that pKr-2 may be a pathogenic factor in PD, and that the inhibition of pKr-2-induced microglial TLR4 may be protective against degeneration of the nigrostriatal DA system in vivo. PMID:26440368

  10. Roles of isolectin B4-binding afferents in colorectal mechanical nociception.

    PubMed

    La, Jun-Ho; Feng, Bin; Kaji, Kaori; Schwartz, Erica S; Gebhart, G F

    2016-02-01

    Isolectin B4-binding (IB4+) dorsal root ganglion (DRG) neurons are distinct from peptidergic DRG neurons in their terminal location in the spinal cord and respective contributions to various classes and modalities of nociception. In DRG neurons innervating the mouse colon (c-DRG neurons), the reported proportion of IB4+ population is inconsistent across studies, and little is known regarding their role in colorectal mechanonociception. To address these issues, in C57BL/6J mice, we quantified IB4+ binding after labeling c-DRG neurons with Fast Blue and examined functional consequences of ablating these neurons by IB4-conjugated saporin. Sixty-one percent of Fast Blue-labeled neurons in the L6 DRG were IB4+, and 95% of these IB4+ c-DRG neurons were peptidergic. Intrathecal administration of IB4-conjugated saporin reduced the proportion of IB4+ c-DRG neurons to 37%, which was due to the loss of c-DRG neurons showing strong to medium IB4+ intensity; c-DRG neurons with weak IB4+ intensity were spared. However, this loss altered neither nociceptive behaviors to colorectal distension nor the relative proportions of stretch-sensitive colorectal afferent classes characterized by single-fiber recordings. These findings demonstrate that more than 1 half of viscerosensory L6 c-DRG neurons in C57BL/6J mouse are IB4+ and suggest, in contrast to the reported roles of IB4+/nonpeptidergic neurons in cutaneous mechanonociception, c-DRG neurons with strong-to-medium IB4+ intensity do not play a significant role in colorectal mechanonociception. PMID:26447707

  11. Early recovery from cow's milk allergy is associated with decreasing IgE and increasing IgG4 binding to cow's milk epitopes

    PubMed Central

    Savilahti, Emma M.; Rantanen, Ville; Lin, Jing S.; Karinen, Sirkku; Saarinen, Kristiina M.; Goldis, Marina; Mäkelä, Mika J.; Hautaniemi, Sampsa; Savilahti, Erkki; Sampson, Hugh

    2010-01-01

    Background Dynamics and balance of allergen specific IgE, IgG4 and IgA binding may contribute to the development of tolerance in cow's milk allergy. Profiling of antibody binding to cow's milk protein epitopes may help in predicting natural history of allergy. Objective To investigate differences in IgE, IgG4 and IgA binding to cow's milk epitopes over time between patients with early recovery or with persisting cow's milk allergy. Methods We studied serum samples at the time of diagnosis (mean age 7 months), one year later and at follow-up (mean age 8.6 years) from 11 patients with persisting IgE-mediated cow's milk allergy at age 8-9 years, and 12 patients who recovered by age 3 years. We measured the binding of IgE, IgG4 and IgA antibodies to sequential epitopes derived from five major cow's milk proteins with a peptide microarray-based immunoassay. We analyzed the data with a novel image processing method together with machine learning prediction. Results IgE epitope binding patterns were stable over time in patients with persisting cow's milk allergy, whereas binding decreased in patients who recovered early. Binding patterns of IgE and IgG4 overlapped. Among patients who recovered early, the signal of IgG4 binding increased while that of IgE decreased over time. IgE and IgG4 binding to a panel of αs1-, αs2-, β-and κ-casein regions predicted outcome with significant accuracy. Conclusions Attaining tolerance to cow's milk is associated with decreased epitope binding by IgE and a concurrent increase in corresponding epitope binding by IgG4. PMID:20462631

  12. Tetranectin-like protein in vertebrate serum: a comparative immunochemical analysis.

    PubMed

    Thougaard, A V; Jaliashvili, I; Christiansen, M

    2001-04-01

    The glycoprotein tetranectin (TN) found in human serum is a 90-kDa homotrimeric C-type lectin binding Ca2+, heparin and plasminogen kringle 4. TN is suggested as being implicated in tissue remodelling. The antigenic reactivity of putative TN was examined in serum from 14 different animal species using three sandwich enzyme immunoassays for human TN. Crab-eating macaque serum showed the strongest reaction, followed by horse and cat. Serum from cow, goat, pig, mouse and chicken reacted weakly, while dog, trout, and the amphibian and the reptile species did not react. The TN-like protein from macaque, horse and cat serum bound heparin and showed the same dependence on Ca2+ for interaction with the monoclonal antibodies as human TN. Gel filtration of sera from the three animal species showed that the TN-like protein eluted as single peaks with a M(r) of 70-90 kDa. Western blotting of horse and cat TN-like protein electrophoresed under reducing conditions showed that the antibodies against human TN reacted with a single band with an approximate M(r) of 30 kDa, indicating that the TN-like protein is also a homotrimer. Horse and cat TN-like protein interacted with human kringle 4-sepharose. Most likely, the reacting protein represents crab-eating macaque, horse and cat homologues of human TN. PMID:11290444

  13. Identification of negative transcriptional factor E4BP4-binding site in the mouse circadian-regulated gene Mdr2.

    PubMed

    Kotaka, Maki; Onishi, Yoshiaki; Ohno, Tomoya; Akaike, Toshihiro; Ishida, Norio

    2008-03-01

    The hepatic transporter Mdr2 is an ATP-binding cassette transporter which excretes phosphatidylcholine into the bile. We showed that the level of Mdr2 mRNA oscillated in circadian fashion in mouse liver whereas such oscillation was dampened in the liver of Clock mutants. To examine transcriptional regulation of the Mdr2 gene we performed luciferase reporter assays using plasmid constructs containing the 5'-flanking region of the Mdr2 gene. Reporter assays using deletion constructs demonstrated that E4BP4 represses the transcriptional activity of the promoter including the D1 and D2 sites within four putative E4BP4-binding sites. Chromatin immunoprecipitation and gel shift assays showed that E4BP4 binds to the D2 site, but not to the D1 site. These data suggested that E4BP4 is a negative transcription factor for circadian Mdr2 mRNA expression. PMID:18242748

  14. CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

    SciTech Connect

    Sterjovski, Jasminka; Churchill, Melissa J.; Roche, Michael; Ellett, Anne; Farrugia, William; Wesselingh, Steven L.; Cunningham, Anthony L.; Ramsland, Paul A.; Gorry, Paul R.

    2011-02-20

    CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.

  15. Inhibition by synthetic peptides from human IgG Fc of OKT4 binding and Fc receptor binding

    SciTech Connect

    Gaarde, W.A.; McClurg, M.R.; Hahn, G.S.; Plummer, J.M.

    1986-03-05

    Synthetic peptides from the Fc region of human IgG were tested for the ability to inhibit IgG rosette formation, /sup 125/I-IgG binding to Fc receptors on lymphocytes, and expression of the T4 cell determinant. The Fc/sub ..gamma../ peptides inhibited IgG rosette formation and competitively inhibited both /sup 125/I-IgG binding to Fc receptors and OKT4 binding to the T4 cell determinant. Peptides containing D-amino acids did not inhibit IgG rosettes, OKT4 binding or /sup 125/I-IgG binding. OKT4 binding to T4 on MNC was inhibited by heat aggregated IgG but not by monomeric IgG. OKT3, OKT8 and OKT11 binding to their determinants was not altered by Fc/sub ..gamma../ peptides, aggregated IgG or monomeric IgG. These results suggest that T4 antigen, Fc receptor for IgG and the Fc/sub ..gamma../ peptide binding site are in close proximity on the cell surface and when occupied by their respective ligands may sterically hinder binding to other sites. Alternatively, Fc/sub ..gamma../ peptides may indirectly regulate cell surface expression of T4 and/or Fc receptors for IgG. These Fc/sub ..gamma../ peptides inhibit the MLR, inhibit antigen induced T cell proliferation and reverse animal models of autoimmune disease. The immunoregulatory activities of these peptides may be related to their selective action on T4 helper/inducer lymphocytes expressing Fc receptors.

  16. Thymic pathogenicity of an HIV-1 envelope is associated with increased CXCR4 binding efficiency and V5-gp41-dependent activity, but not V1/V2-associated CD4 binding efficiency and viral entry

    SciTech Connect

    Meissner, Eric G.; Coffield, Vernon M.; Su Lishan . E-mail: lsu@med.unc.edu

    2005-06-05

    We previously described a thymus-tropic HIV-1 envelope (R3A Env) from a rapid progressor obtained at the time of transmission. An HIV-1 molecular recombinant with the R3A Env supported extensive replication and pathogenesis in the thymus and did not require Nef. Another Env from the same patient did not display the same thymus-tropic pathogenesis (R3B Env). Here, we show that relative to R3B Env, R3A Env enhances viral entry of T cells, increases fusion-induced cytopathicity, and shows elevated binding efficiency for both CD4 and CXCR4, but not CCR5, in vitro. We created chimeric envelopes to determine the region(s) responsible for each in vitro phenotype and for thymic pathogenesis. Surprisingly, while V1/V2 contributed to enhanced viral entry, CD4 binding efficiency, and cytopathicity in vitro, it made no contribution to thymic pathogenesis. Rather, CXCR4 binding efficiency and V5-gp41-associated activity appear to independently contribute to thymic pathogenesis of the R3A Env. These data highlight the contribution of unique HIV pathogenic factors in the thymic microenvironment and suggest that novel mechanisms may be involved in Env pathogenic activity in vivo.

  17. Seeking for binding determinants of the prion protein to human plasminogen

    NASA Astrophysics Data System (ADS)

    Menziani, M. C.; de Benedetti, P. G.; Langella, E.; Barone, V.

    Plasminogen (Pg), a pro-protease implicated in neuronal excitotoxicity, has recently been identified as binding to prion protein (PrP) from several species. Although the precise effect of the binding of PrP to plasminogen in the course of prion-caused diseases has not yet been demonstrated, the implications of this important finding for diagnostic applications are straightforward. In this paper we have investigated the possible modes of interaction of PrP with plasminogen, by means of molecular modelling and computational simulation techniques. To this goal, we first exploited the information available for the mK2Pg/VEK-30 complex in order to identify the PrP residues which satisfy the specific electronic and geometric requirements needed to interact with the kringle lysine binding site, we compared the relevant mK2Pg/VEK-30 and mK2Pg/PrP interactions obtained from the simulated protein-protein complexes and we assessed the docking hypothesis utilized for the mK2Pg/PrP complex by simulating the interaction of PrP with the multi-kringle angiostatin, a more realistic model of the physiological target. The results obtained will be instrumental for planning experiments tailored to clarify the role of the plasminogen activator system in prion-related diseases and, eventually, for mimicking dominant binding determinants through structure-based drug design.

  18. Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site

    PubMed Central

    Crooks, Ema T.; Tong, Tommy; Chakrabarti, Bimal; Narayan, Kristin; Georgiev, Ivelin S.; Menis, Sergey; Huang, Xiaoxing; Kulp, Daniel; Osawa, Keiko; Muranaka, Janelle; Stewart-Jones, Guillaume; Destefano, Joanne; O’Dell, Sijy; LaBranche, Celia; Robinson, James E.; Montefiori, David C.; McKee, Krisha; Du, Sean X.; Doria-Rose, Nicole; Kwong, Peter D.; Mascola, John R.; Zhu, Ping; Schief, William R.; Wyatt, Richard T.; Whalen, Robert G.; Binley, James M.

    2015-01-01

    Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine. PMID:26023780

  19. Relationship of the human immunodeficiency virus type 1 gp120 third variable loop to a component of the CD4 binding site in the fourth conserved region.

    PubMed Central

    Wyatt, R; Thali, M; Tilley, S; Pinter, A; Posner, M; Ho, D; Robinson, J; Sodroski, J

    1992-01-01

    Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions. Images PMID:1279195

  20. Envelope residue 375 substitutions in simian–human immunodeficiency viruses enhance CD4 binding and replication in rhesus macaques

    PubMed Central

    Li, Hui; Wang, Shuyi; Kong, Rui; Ding, Wenge; Lee, Fang-Hua; Parker, Zahra; Kim, Eunlim; Learn, Gerald H.; Hahn, Paul; Policicchio, Ben; Brocca-Cofano, Egidio; Deleage, Claire; Hao, Xingpei; Chuang, Gwo-Yu; Gorman, Jason; Gardner, Matthew; Lewis, Mark G.; Hatziioannou, Theodora; Santra, Sampa; Apetrei, Cristian; Pandrea, Ivona; Alam, S. Munir; Liao, Hua-Xin; Shen, Xiaoying; Tomaras, Georgia D.; Farzan, Michael; Chertova, Elena; Keele, Brandon F.; Estes, Jacob D.; Lifson, Jeffrey D.; Doms, Robert W.; Montefiori, David C.; Haynes, Barton F.; Sodroski, Joseph G.; Kwong, Peter D.; Hahn, Beatrice H.; Shaw, George M.

    2016-01-01

    Most simian–human immunodeficiency viruses (SHIVs) bearing envelope (Env) glycoproteins from primary HIV-1 strains fail to infect rhesus macaques (RMs). We hypothesized that inefficient Env binding to rhesus CD4 (rhCD4) limits virus entry and replication and could be enhanced by substituting naturally occurring simian immunodeficiency virus Env residues at position 375, which resides at a critical location in the CD4-binding pocket and is under strong positive evolutionary pressure across the broad spectrum of primate lentiviruses. SHIVs containing primary or transmitted/founder HIV-1 subtype A, B, C, or D Envs with genotypic variants at residue 375 were constructed and analyzed in vitro and in vivo. Bulky hydrophobic or basic amino acids substituted for serine-375 enhanced Env affinity for rhCD4, virus entry into cells bearing rhCD4, and virus replication in primary rhCD4 T cells without appreciably affecting antigenicity or antibody-mediated neutralization sensitivity. Twenty-four RMs inoculated with subtype A, B, C, or D SHIVs all became productively infected with different Env375 variants—S, M, Y, H, W, or F—that were differentially selected in different Env backbones. Notably, SHIVs replicated persistently at titers comparable to HIV-1 in humans and elicited autologous neutralizing antibody responses typical of HIV-1. Seven animals succumbed to AIDS. These findings identify Env–rhCD4 binding as a critical determinant for productive SHIV infection in RMs and validate a novel and generalizable strategy for constructing SHIVs with Env glycoproteins of interest, including those that in humans elicit broadly neutralizing antibodies or bind particular Ig germ-line B-cell receptors. PMID:27247400

  1. Envelope residue 375 substitutions in simian-human immunodeficiency viruses enhance CD4 binding and replication in rhesus macaques.

    PubMed

    Li, Hui; Wang, Shuyi; Kong, Rui; Ding, Wenge; Lee, Fang-Hua; Parker, Zahra; Kim, Eunlim; Learn, Gerald H; Hahn, Paul; Policicchio, Ben; Brocca-Cofano, Egidio; Deleage, Claire; Hao, Xingpei; Chuang, Gwo-Yu; Gorman, Jason; Gardner, Matthew; Lewis, Mark G; Hatziioannou, Theodora; Santra, Sampa; Apetrei, Cristian; Pandrea, Ivona; Alam, S Munir; Liao, Hua-Xin; Shen, Xiaoying; Tomaras, Georgia D; Farzan, Michael; Chertova, Elena; Keele, Brandon F; Estes, Jacob D; Lifson, Jeffrey D; Doms, Robert W; Montefiori, David C; Haynes, Barton F; Sodroski, Joseph G; Kwong, Peter D; Hahn, Beatrice H; Shaw, George M

    2016-06-14

    Most simian-human immunodeficiency viruses (SHIVs) bearing envelope (Env) glycoproteins from primary HIV-1 strains fail to infect rhesus macaques (RMs). We hypothesized that inefficient Env binding to rhesus CD4 (rhCD4) limits virus entry and replication and could be enhanced by substituting naturally occurring simian immunodeficiency virus Env residues at position 375, which resides at a critical location in the CD4-binding pocket and is under strong positive evolutionary pressure across the broad spectrum of primate lentiviruses. SHIVs containing primary or transmitted/founder HIV-1 subtype A, B, C, or D Envs with genotypic variants at residue 375 were constructed and analyzed in vitro and in vivo. Bulky hydrophobic or basic amino acids substituted for serine-375 enhanced Env affinity for rhCD4, virus entry into cells bearing rhCD4, and virus replication in primary rhCD4 T cells without appreciably affecting antigenicity or antibody-mediated neutralization sensitivity. Twenty-four RMs inoculated with subtype A, B, C, or D SHIVs all became productively infected with different Env375 variants-S, M, Y, H, W, or F-that were differentially selected in different Env backbones. Notably, SHIVs replicated persistently at titers comparable to HIV-1 in humans and elicited autologous neutralizing antibody responses typical of HIV-1. Seven animals succumbed to AIDS. These findings identify Env-rhCD4 binding as a critical determinant for productive SHIV infection in RMs and validate a novel and generalizable strategy for constructing SHIVs with Env glycoproteins of interest, including those that in humans elicit broadly neutralizing antibodies or bind particular Ig germ-line B-cell receptors. PMID:27247400

  2. IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons

    PubMed Central

    Harrison, Benjamin J.; Flight, Robert M.; Gomes, Cynthia; Venkat, Gayathri; Ellis, Steven R; Sankar, Uma; Twiss, Jeffery L.; Rouchka, Eric C.; Petruska, Jeffrey C.

    2013-01-01

    Calcium/Calmodulin-dependent protein Kinase 4 (Gene and transcript: CaMK4; Protein: CaMKIV) is the nuclear effector of the Ca2+/Calmodulin Kinase (CaMK) pathway where it co-ordinates transcriptional responses. However, CaMKIV is present in the cytoplasm and axons of subpopulations of neurons, including some sensory neurons of the dorsal root ganglia (DRG), suggesting an extra-nuclear role for this protein. We observed that CaMKIV was expressed strongly in the cytoplasm and axons of a subpopulation of small diameter DRG neurons, most likely cutaneous nociceptors by virtue of their binding the isolectin IB4. In IB4+ spinal nerve axons, 20% of CaMKIV was co-localized with the endocytic marker Rab7 in axons that highly expressed CAM-Kinase-Kinase (CAMKK), an upstream activator of CaMKIV, suggesting a role for CaMKIV in signalling though signalling endosomes. Using fluorescent in situ hybridization (FISH) with riboprobes, we also observed that small diameter neurons expressed high levels of a novel 3' untranslated region (UTR) variant of CaMK4 mRNA. Using rapid amplification of cDNA ends (RACE), RT-PCR with gene-specific primers, and cDNA sequencing analyses we determined that the novel transcript contains an additional 10kb beyond the annotated gene terminus to a highly conserved alternate poly-adenylation site. qPCR analyses of fluorescent-activated cell sorted (FACS) DRG neurons confirmed that this 3'UTR-extended variant was preferentially expressed in IB4-binding neurons. Computational analyses of the 3'-UTR sequence predict that UTR-extension introduces consensus sites for RNA-binding proteins (RBPs) including the Embryonic Lethal Abnormal Vision (ELAV)/Hu family proteins. We consider the possible implications of axonal CaMKIV in the context of the unique properties of IB4-binding DRG neurons. PMID:23817991

  3. Design, synthesis and antiviral activity of entry inhibitors that target the CD4-binding site of HIV-1

    PubMed Central

    Curreli, Francesca; Choudhury, Spreeha; Pyatkin, Ilya; Zagorodnikov, Victor P.; Bulay, Anna Khulianova; Altieri, Andrea; Kwon, Young Do; Kwong, Peter D.; Debnath, Asim K.

    2012-01-01

    The CD4 binding site on HIV-1 gp120 has been validated as a drug target to prevent HIV-1 entry to cells. Previously, we identified two small molecule inhibitors consisting of a 2,2,6,6-tetramethylpiperidine ring linked by an oxalamide to a p-halide-substituted phenyl group, which target this site, specifically, a cavity termed “Phe43 cavity”. Here we use synthetic chemistry, functional assessment and structure-based analysis to explore variants of each region of these inhibitors for improved antiviral properties. Alterations of the phenyl group and of the oxalamide linker indicated that these regions were close to optimal in the original lead compounds. Design of a series of compounds, where the tetramethylpiperidine ring was replaced with new scaffolds, lead to improved antiviral activity. These new scaffolds provide insight into the surface chemistry at the entrance of the cavity and offer additional opportunities by which to optimize further these potential-next-generation therapeutics and microbicides against HIV-1. PMID:22524483

  4. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

    SciTech Connect

    Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; Amos, Joshua  D.; Kumar, Amit; Hora, Bhavna; Marshall, Dawn  J.; Whitesides, John  F.; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey  E.; Hwang, Kwan-Ki; Lu, Xiaozhi; Bonsignori, Mattia; Finzi, Andrés; Vandergrift, Nathan  A.; Alam, S.  Munir; Ferrari, Guido; Shen, Xiaoying; Tomaras, Georgia  D.; Kamanga, Gift; Cohen, Myron  S.; Sam, Noel  E.; Kapiga, Saidi; Gray, Elin S.; Tumba, Nancy  L.; Morris, Lynn; Zolla-Pazner, Susan; Gorny, Miroslaw  K.; Mascola, John  R.; Hahn, Beatrice H.; Shaw, George  M.; Sodroski, Joseph  G.; Liao, Hua-Xin; Montefiori, David C.; Hraber, Peter T.; Korber, Bette T.; Haynes, Barton F.

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.

  5. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

    DOE PAGESBeta

    Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; Amos, Joshua  D.; Kumar, Amit; Hora, Bhavna; Marshall, Dawn  J.; Whitesides, John  F.; Xia, Shi-Mao; Parks, Robert; et al

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tiermore » 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.« less

  6. A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo

    PubMed Central

    Freund, Natalia T.; Horwitz, Joshua A.; Nogueira, Lilian; Sievers, Stuart A.; Scharf, Louise; Scheid, Johannes F.; Gazumyan, Anna; Liu, Cassie; Velinzon, Klara; Goldenthal, Ariel; Sanders, Rogier W.; Moore, John P.; Bjorkman, Pamela J.; Seaman, Michael S.; Walker, Bruce D.; Klein, Florian; Nussenzweig, Michel C.

    2015-01-01

    The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC80 value of 0.42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM.bl assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans. PMID:26516768

  7. Uniform {sup 15}N- and {sup 15}N/{sup 13}C-labeling of proteins in mammalian cells and solution structure of the amino terminal fragment of u-PA

    SciTech Connect

    Hansen, A.P.; Petros, A.M.; Meadows, R.P.; Mazar, A.P.; Nettesheim, D.G.; Pederson, T.M.; Fesik, S.W.

    1994-12-01

    Urokinase-type plasminogen activator (u-PA) is a 54-kDa glycoprotein that catalyzes the conversion of plasminogen to plasmin, a broad-specificity protease responsible for the degradation of fibrin clots and extracellular matrix components. The u-PA protein consists of three individual modules: a growth factor domain (GFD), a kringle, and a serine protease domain. The amino terminal fragment (ATF) includes the GFD-responsible for u-PA binding to its receptor-and the kringle domains. This protein was expressed and uniformly {sup 15}N-and {sup 15}N/{sup 13}C-labeled in mammalian cells by methods that will be described. In addition, we present the three-dimensional structure of ATF that was derived from 1299 NOE-derived distance restraints along with the {phi} angle and hydrogen bonding restraints. Although the individual domains in the structures were highly converged, the two domains are structurally independent. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor domain of u-PA and other homologous proteins were observed in the region that has been implicated in binding the urokinase receptor. These results may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

  8. (/sup 3/H)leukotriene B/sub 4/ binding to the guinea pig spleen membranes: a rich tissue source for a high affinity leukotriene B/sub 4/ receptor site

    SciTech Connect

    Cheng, J.B.; Kohi, F.; Townley, R.G.

    1986-03-05

    To select a tissue rich for the high affinity leukotriene (LT)B/sub 4/ receptor site, they compared binding of 1 nM (/sup 3/H)LTB/sub 4/ (180 Ci/mmol) to the crude membrane preparations of guinea pig spleen, thymus, lung, uterus, bladder, brain, adrenal gland, small intestine, liver, kidney and heart. They found that the membrane preparations from spleen contained the highest binding activity per mg protein. They characterized the LTB/sub 4/ binding to the spleen preparation in detail. LTB/sub 4/ binding was rapid, reversible, stereoselective and saturable. The data from equilibrium experiments showed a linear Scatchard plot with a K/sub d/ of 1.6 nM and a binding site density of 259 fmol/mg prot. The rank order of agents competing for spleen (/sup 3/H)LTB/sub 4/ binding at 25/sup 0/C was: LTB/sub 4/ (K/sub i/ = 2.8 nM) > 20-OH-LTB/sub 4/ (23 nM) > LTA/sub 4/ (48 nM) > LTA/sub 4/ methyl ester (0.13 ..mu..M) > 20-COOH-LTB/sub 4/ (> 6.6 ..mu..M) greater than or equal to arachidonic acid (0.15 mM) similarly ordered FPL-55,712 (0.11 mM). At 4/sup 0/C, LTB/sub 4/ (2.3 nM) competed at least 10x more effectively than 20-OH-LTB/sub 4/ (29 nM) and 20-COOH-LTB/sub 4/ (> 6.6 ..mu..M). HPLC analysis indicated that incubation of 84 ng LTB/sub 4/ with the spleen membrane at 25/sup 0/C did not result in the formation of 20-OH-LTB/sub 4/ (< 1 ng) in the filtrate. They conclude that guinea pig spleen contains a rich tissue source of high affinity (/sup 3/H)LTB/sub 4/ receptor binding sites.

  9. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  10. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  11. Structure of KRT4 binding domain of Srr-1 from Streptococcus agalactiae reveals a novel β-sheet complementation.

    PubMed

    Sundaresan, Ramya; Samen, Ulrike; Ponnuraj, Karthe

    2015-04-01

    The serine rich repeat protein-1 (Srr-1) is an adhesive protein of Streptococcus agalactiae. It is the first bacterial protein identified to interact with human keratin 4 (K4 or KRT4). Within Srr-1, the residues 311-641 constitute the non-repeat ligand binding region (Srr-1-BR(311-641)). The C-terminal part of Srr-1-BR(311-641), comprising of residues 485-642 (termed Srr-1-K4BD), have been identified to bind to K4. Here we report the crystal structure of recombinant Srr-1-K4BD(485-642) and its possible mode of interaction with K4 through docking studies. The dimeric structure of Srr-1-K4BD(485-642) reveals a novel two way "slide lock" parallel β-sheet complementation where the C-terminal strand of one monomer is positioned anti-parallel to the N-terminal strand of the adjacent monomer and this arrangement is not seen so far in any of the homologous structures. The dimerization of Srr-1-K4BD(485-642) observed both in the crystal structure and in solution suggests that similar domain association could also be possible in in vivo and we propose this association would likely generate a new binding site for another host molecule. It is likely that the adhesin can recognize multiple ligands using its ligand binding sub-domains through their intra and inter domain association with one another. PMID:25603146

  12. Generation and characterization of monoclonal antibodies to the putative CD4-binding domain of human immunodeficiency virus type 1 gp120.

    PubMed Central

    Sun, N C; Ho, D D; Sun, C R; Liou, R S; Gordon, W; Fung, M S; Li, X L; Ting, R C; Lee, T H; Chang, N T

    1989-01-01

    A panel of seven monoclonal antibodies against the relatively conserved CD4-binding domain on human immunodeficiency virus type 1 (HIV-1) gp120 was generated by immunizing mice with purified gp120. These monoclonal antibodies reacted specifically with gp120 in an enzyme-linked immunosorbent assay and Western blots (immunoblots). By using synthetic peptides as antigens in the immunosorbent assay, the epitopes of these seven monoclonal antibodies were mapped to amino acid residues 423 to 437 of gp120. Further studies with radioimmunoprecipitation assays showed that they cross-reacted with both gp120 and gp160 of diverse HIV-1 isolates (HTLV-IIIB, HTLV-IIIRF, HTLV-IIIAL, and HTLV-IIIWMJ). They also bound specifically to H9 cells infected with HTLV-IIIB, HTLV-IIIRF, HTLV-IIIAL, HTLV-IIIZ84, and HTLV-IIIZ34 in indirect immunofluorescence studies. In addition, they blocked effectively the binding of HIV-1 to CD4+ C8166 cells. Despite the similarity of these properties, the monoclonal antibodies differed in neutralizing activity against HTLV-IIIB, HTLV-IIIRF, and HTLV-IIIAL, as demonstrated in both syncytium-forming assays and infectivity assays. Our findings suggest that these group-specific monoclonal antibodies to the putative CD4-binding domain on gp120 are potential candidates for development of therapeutic agents against acquired immunodeficiency disease syndrome. PMID:2474670

  13. Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE.

    PubMed

    Moradi-Fard, Sarah; Sarthi, Jessica; Tittel-Elmer, Mireille; Lalonde, Maxime; Cusanelli, Emilio; Chartrand, Pascal; Cobb, Jennifer A

    2016-08-01

    SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, nse3-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres. Nse3 interacts physically and genetically with two Rap1-binding factors, Rif2 and Sir4. Reduction in telomere-associated Smc5/6 leads to defects in telomere clustering, dispersion of the silencing factor, Sir4, and a loss in transcriptional repression for sub-telomeric genes and non-coding telomeric repeat-containing RNA (TERRA). SIR4 recovery at telomeres is reduced in cells lacking Smc5/6 functionality and vice versa. However, nse3-1/ sir4 Δ double mutants show additive defects for telomere shortening and TPE indicating the contribution of Smc5/6 to telomere homeostasis is only in partial overlap with SIR factor silencing. These findings support a role for Smc5/6 in telomere maintenance that is separate from its canonical role(s) in HR-mediated events during replication and telomere elongation. PMID:27564449

  14. Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE

    PubMed Central

    Chartrand, Pascal; Cobb, Jennifer A.

    2016-01-01

    SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, nse3-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres. Nse3 interacts physically and genetically with two Rap1-binding factors, Rif2 and Sir4. Reduction in telomere-associated Smc5/6 leads to defects in telomere clustering, dispersion of the silencing factor, Sir4, and a loss in transcriptional repression for sub-telomeric genes and non-coding telomeric repeat-containing RNA (TERRA). SIR4 recovery at telomeres is reduced in cells lacking Smc5/6 functionality and vice versa. However, nse3-1/ sir4 Δ double mutants show additive defects for telomere shortening and TPE indicating the contribution of Smc5/6 to telomere homeostasis is only in partial overlap with SIR factor silencing. These findings support a role for Smc5/6 in telomere maintenance that is separate from its canonical role(s) in HR-mediated events during replication and telomere elongation. PMID:27564449

  15. Lubricin/Proteoglycan 4 Binding to CD44 Receptor: A Mechanism of Lubricin’s suppression of Pro-inflammatory Cytokine Induced Synoviocyte Proliferation

    PubMed Central

    Al-Sharif, Afnan; Jamal, Maha; Zhang, Ling; Larson, Katherine; Schmidt, Tannin; Jay, Gregory; Elsaid, Khaled

    2015-01-01

    Objective To evaluate recombinant human proteoglycan 4 (rhPRG4) binding to CD44 receptor and its consequence on cytokine induced synoviocyte proliferation. Methods rhPRG4 binding to CD44 and competition with high molecular weight hyaluronic acid (HMW HA) was evaluated using a direct enzyme linked immunosorbent assay (ELISA) and surface plasmon resonance. Sialidase-A and O-glycosidase digestion of rhPRG4 was performed and CD44 binding was evaluated using ELISA. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were stimulated with interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α) for 48 hours in the presence or absence of rhPRG4 or HMW HA at 20, 40 and 80μg/ml and cell proliferation was measured. CD44 contribution was assessed by co-incubation with a CD44 antibody (IM7). The anti-proliferative effect of rhPRG4 was investigated following treatment of Prg4−/− synoviocytes with IL-1β or TNF-α in the presence or absence of IM7. Results rhPRG4 binds CD44 and interferes with HMW HA CD44 binding. Removal of sialic acid and O-glycosylations significantly increased CD44 binding by rhPRG4 (p<0.001). rhPRG4 and HMW HA at 40 and 80μg/ml significantly suppressed IL-1β induced RA-FLS proliferation (p<0.05). rhPRG4 at 20, 40 and 80μg/ml significantly suppressed TNF-α induced RA-FLS proliferation (p<0.05). CD44 neutralization reversed the effect of rhPRG4 on IL-1β and TNF-α stimulated RA-FLS and the effect of HMW HA on IL-1β stimulated RA-FLS. rhPRG4 inhibited cytokine-induced proliferation of Prg4−/− synoviocytes which could be prevented by blocking CD44. Conclusion Lubricin is a novel putative ligand for CD44 and may control synoviocyte overgrowth in inflammatory arthropathies via a CD44-mediated mechanism. PMID:25708025

  16. Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans

    PubMed Central

    Julien, Jean-Philippe; Sok, Devin; Khayat, Reza; Lee, Jeong Hyun; Doores, Katie J.; Walker, Laura M.; Ramos, Alejandra; Diwanji, Devan C.; Pejchal, Robert; Cupo, Albert; Katpally, Umesh; Depetris, Rafael S.; Stanfield, Robyn L.; McBride, Ryan; Marozsan, Andre J.; Paulson, James C.; Sanders, Rogier W.; Moore, John P.; Burton, Dennis R.; Poignard, Pascal; Ward, Andrew B.; Wilson, Ian A.

    2013-01-01

    New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml−1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained. PMID:23658524

  17. Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.

    PubMed

    Julien, Jean-Philippe; Sok, Devin; Khayat, Reza; Lee, Jeong Hyun; Doores, Katie J; Walker, Laura M; Ramos, Alejandra; Diwanji, Devan C; Pejchal, Robert; Cupo, Albert; Katpally, Umesh; Depetris, Rafael S; Stanfield, Robyn L; McBride, Ryan; Marozsan, Andre J; Paulson, James C; Sanders, Rogier W; Moore, John P; Burton, Dennis R; Poignard, Pascal; Ward, Andrew B; Wilson, Ian A

    2013-01-01

    New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1). Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained. PMID:23658524

  18. The extracellular matrix proteins laminin and fibronectin contain binding domains for human plasminogen and tissue plasminogen activator.

    PubMed

    Moser, T L; Enghild, J J; Pizzo, S V; Stack, M S

    1993-09-01

    This study describes the binding of plasminogen and tissue-type plasminogen activator (t-PA) to the extracellular matrix proteins fibronectin and laminin. Plasminogen bound specifically and saturably to both fibronectin and laminin immobilized on microtiter wells, with Kd(app) values of 115 and 18 nM, respectively. Limited proteolysis by endoproteinase V8 coupled with ligand blotting analysis showed that both plasminogen and t-PA preferentially bind to a 55-kDa fibronectin fragment and a 38-kDa laminin fragment. Amino acid sequence analysis demonstrated that the 5-kDa fragment originates with the fibronectin amino terminus whereas the laminin fragment was derived from the carboxyl-terminal globular domain of the laminin A chain. Ligand blotting experiments using isolated plasminogen domains were also used to identify distinct regions of the plasminogen molecule involved in fibronectin and laminin binding. Solution phase fibronectin binding to immobilized plasminogen was mediated primarily via lysine binding site-dependent interactions with plasminogen kringles 1-4. Lysine binding site-dependent binding of soluble laminin to immobilized plasminogen kringles 1-5 as well as an additional lysine binding site-independent interaction between mini-plasminogen and the 38-kDa laminin A chain fragment were also observed. These studies demonstrate binding of plasminogen and tissue-type plasminogen activator to specific regions of the extracellular matrix glycoproteins laminin and fibronectin and provide further insight into the mechanism of regulation of plasminogen activation by components of the extracellular matrix. PMID:8360181

  19. Molecular Interactions of Human Plasminogen with Fibronectin-binding Protein B (FnBPB), a Fibrinogen/Fibronectin-binding Protein from Staphylococcus aureus.

    PubMed

    Pietrocola, Giampiero; Nobile, Giulia; Gianotti, Valentina; Zapotoczna, Marta; Foster, Timothy J; Geoghegan, Joan A; Speziale, Pietro

    2016-08-26

    Staphylococcus aureus is a commensal bacterium that has the ability to cause superficial and deep-seated infections. Like several other invasive pathogens, S. aureus can capture plasminogen from the human host where it can be converted to plasmin by host plasminogen activators or by endogenously expressed staphylokinase. This study demonstrates that sortase-anchored cell wall-associated proteins are responsible for capturing the bulk of bound plasminogen. Two cell wall-associated proteins, the fibrinogen- and fibronectin-binding proteins A and B, were found to bind plasminogen, and one of them, FnBPB, was studied in detail. Plasminogen captured on the surface of S. aureus- or Lactococcus lactis-expressing FnBPB could be activated to the potent serine protease plasmin by staphylokinase and tissue plasminogen activator. Plasminogen bound to recombinant FnBPB with a KD of 0.532 μm as determined by surface plasmon resonance. Plasminogen binding did not to occur by the same mechanism through which FnBPB binds to fibrinogen. Indeed, FnBPB could bind both ligands simultaneously indicating that their binding sites do not overlap. The N3 subdomain of FnBPB contains the full plasminogen-binding site, and this includes, at least in part, two conserved patches of surface-located lysine residues that were recognized by kringle 4 of the host protein. PMID:27387503

  20. Impact of Nucleotide Mutations at the HNF3- and HNF4-Binding Sites in Enhancer 1 on Viral Replication in Patients with Chronic Hepatitis B Virus Infection

    PubMed Central

    Cho, Eun-young; Kim, Hyung-jin; Park, Channy; So, Hong-seob; Park, Rae Kil

    2013-01-01

    Background/Aims The hepatitis B virus (HBV) genome contains binding sites for hepatocyte nuclear factors (HNF) 3 and 4 in the core domain of enhancer 1 (Enh1), and mutations in this domain have a strong impact on virus replication. We aimed to identify frequent base-mutation sites in the core domain of Enh1 and to examine the impact of these mutations on viral replication. Methods We studied virological characteristics and genetic sequences in 387 patients with chronic hepatitis B. We evaluated functional differences associated with specific mutations within the core domain of Enh1. Results Mutations in the core domain were found with significant frequency in C1126 (122/387 [31.5%], the binding site for HNF3) and in C1134 (106/387 [27.4%], the binding site for HNF4). A single mutation at nt 1126 (C1126) was identified in 17/123 (13.8%), and 105/123 (85.4%) had double mutations (C1126/1134). The level of HBV DNA (log10 copies/mL) was lower in single mutants (C1126, 5.81±1.25) than in wild (6.80±1.65) and double mutants (C1126/1134, 6.81±1.54). Similarly, the relative luciferase activity of C1126 and C1126/C1134 was 0.18 and 1.12 times that of the wild-type virus, respectively. Conclusions Mutations in the HNF3 binding site inhibit viral replication, whereas mutations at the HNF4 binding site restore viral replication. PMID:24073315

  1. In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120

    PubMed Central

    Visciano, Maria Luisa; Tuen, Michael; Gorny, Miroslaw K.; Hioe, Catarina E.

    2008-01-01

    The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus. Moreover, mice immunized with the gp120/anti-CD4bs mAb complexes produced higher titers of gp120-specific serum IgG and IgA than mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced antibody production was directed against V3 and correlated with better exposure of V3 on the gp120/anti-CD4bs mAb complexes. The higher antibody reactivity was evident against the homologous V3LAI peptide, but not against heterologous V3 peptides. Potent neutralization activity against HIV-1LAI was also observed in the sera from mice immunized with gp120/anti-CD4bs mAb complexes, although the sera exhibited poor neutralizing activities against other viruses tested. These results indicate that the anti-CD4bs antibodies alter the antigenicity and immunogenicity of gp120, leading to enhanced production of anti-gp120 antibodies directed particularly against the V3 region. PMID:18054978

  2. BBA70 of Borrelia burgdorferi Is a Novel Plasminogen-binding Protein*

    PubMed Central

    Koenigs, Arno; Hammerschmidt, Claudia; Jutras, Brandon L.; Pogoryelov, Denys; Barthel, Diana; Skerka, Christine; Kugelstadt, Dominik; Wallich, Reinhard; Stevenson, Brian; Zipfel, Peter F.; Kraiczy, Peter

    2013-01-01

    The Lyme disease spirochete Borrelia burgdorferi lacks endogenous, surface-exposed proteases. In order to efficiently disseminate throughout the host and penetrate tissue barriers, borreliae rely on recruitment of host proteases, such as plasmin(ogen). Here we report the identification of a novel plasminogen-binding protein, BBA70. Binding of plasminogen is dose-dependent and is affected by ionic strength. The BBA70-plasminogen interaction is mediated by lysine residues, primarily located in a putative C-terminal α-helix of BBA70. These lysine residues appear to interact with the lysine-binding sites in plasminogen kringle domain 4 because a deletion mutant of plasminogen lacking that domain was unable to bind to BBA70. Bound to BBA70, plasminogen activated by urokinase-type plasminogen activator was able to degrade both a synthetic chromogenic substrate and the natural substrate fibrinogen. Furthermore, BBA70-bound plasmin was able to degrade the central complement proteins C3b and C5 and inhibited the bacteriolytic effects of complement. Consistent with these functional activities, BBA70 is located on the borrelial outer surface. Additionally, serological evidence demonstrated that BBA70 is produced during mammalian infection. Taken together, recruitment and activation of plasminogen could play a beneficial role in dissemination of B. burgdorferi in the human host and may possibly aid the spirochete in escaping the defense mechanisms of innate immunity. PMID:23861404

  3. Competition for in vitro (/sup 3/H)gibberellin A/sub 4/ binding in cucumber by gibberellins and their derivatives. [Cucumis sativus L. cv National Pickling

    SciTech Connect

    Yalpani, N.; Srivastava, L.M.

    1985-12-01

    The gibberellin (GA) binding properties of a cytosol fraction from hypocotyls of cucumber (Cucumis sativus L. cv National Pickling) were examined using a DEAE filter paper assay, (/sup 3/H)GA/sub 4/, and over 20 GAs, GA derivatives and other growth regulators. The results demonstrate structural specificity of the binding protein for ..gamma..-lactonic C-19 GAs with a 3 ..beta..-hydroxyl and a C-6 carboxyl group. Additional hydroxylations of the A, C, or D ring of the ent-gibberellane skeleton and methylation of the C-6 carboxyl impede or abolish binding affinity. Bioassay data are generally supported by the in vitro results but significantly GA/sub 9/ and GA/sub 36/, both considered to be precursors of GA/sub 4/ in cucumber, show no affinity for the binding protein. The results are discussed in relation to the active site of the putative GA/sub 4/ receptor in cucumber.

  4. Thermodynamic and Kinetics Analysis of Peptides Derived from CapZ, NDR, p53, HDM2, and HDM4 Binding to Human S100B

    PubMed Central

    Wafer, Lucas N.; Streicher, Werner W.; McCallum, Scott A.; Makhatadze, George I.

    2012-01-01

    S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer's and Parkinson's disease. Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2 and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, these peptides were screened against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone 1H and 15N were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic Kon rate constants observed in this study, Kon ≥1×107 M-1s-1, suggest that S100B utilizes a “fly casting mechanism” in the recognition of these peptide targets. PMID:22913742

  5. Slow formation of stable complexes during coincubation of a minimal rRNA and ribosomal protein S4

    PubMed Central

    Mayerle, Megan; Bellur, Deepti L.; Woodson, Sarah A.

    2011-01-01

    Ribosomal protein S4 binds and stabilizes a five-helix junction in the 5’ domain of the 16S rRNA, and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and the five-helix junction reorganize their structures. We show that labile S4 complexes rearrange to stable complexes within a few minutes at 42°C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show this structural change depends only on 16S residues within the S4 binding site. SHAPE chemical-probing experiments showed that S4 strongly stabilizes the five-helix junction and helix 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in helix 16 that makes specific contacts with the S4 N-terminal extension, and a right angle motif between helices 3, 4 and 18, require a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of helix 3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation. PMID:21821049

  6. Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells.

    PubMed

    Liang, C L; Tsai, C N; Chung, P J; Chen, J L; Sun, C M; Chen, R H; Hong, J H; Chang, Y S

    2000-11-10

    In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells. PMID:11062049

  7. CD4 peptide-protein conjugates, but not recombinant human CD4, bind to recombinant gp120 from the human immunodeficiency virus in the presence of serum from AIDS patients.

    PubMed Central

    Ghetie, V; Slaughter, C; Wheeler, H T; Uhr, J W; Vitetta, E S

    1991-01-01

    Sera from human immunodeficiency virus-positive (HIV+; Walter Reed stage 6) individuals inhibit the interaction between recombinant human CD4 and recombinant gp120 from HIV (rCD4 and rgp120, respectively), thereby interfering with the ability of soluble rCD4 to block infection with HIV or rCD4-toxin conjugates to kill HIV-infected cells. In this report we demonstrate that the inhibitory activity of such sera is caused primarily by anti-gp120 antibodies that do not recognize the CD4 interaction site on gp120. To circumvent the problem of inhibition, we have generated a construct containing a peptide of CD4 (residues 41-84) conjugated to ovalbumin (three to five peptides per molecule). This multivalent conjugate binds to rgp120 and binding is not inhibited by antibodies in HIV+ sera. PMID:2062847

  8. Antitumor effects of non-replicative herpes simplex vectors expressing antiangiogenic proteins and thymidine kinase on Lewis lung carcinoma establishment and growth.

    PubMed

    Berto, E; Bozac, A; Volpi, I; Lanzoni, I; Vasquez, F; Melara, N; Manservigi, R; Marconi, P

    2007-09-01

    There is growing evidence that combinations of antiangiogenic proteins with other antineoplastic treatments such as chemo- or radiotherapy and suicide genes-mediated tumor cytotoxicity lead to synergistic effects. In the present work, we tested the activity of two non-replicative herpes simplex virus (HSV)-1-based vectors, encoding human endostatin::angiostatin or endostatin::kringle5 fusion proteins in combination with HSV-1 thymidine kinase (TK) molecule, on endothelial cells (ECs) and Lewis lung carcinoma (LLC) cells. We observed a significant reduction of the in vitro growth, migration and tube formation by primary ECs upon direct infection with the two recombinant vectors or cultivation with conditioned media obtained from the vector-infected LLC cells. Moreover, direct cytotoxic effect of HSV-1 TK on both LLC and ECs was demonstrated. We then tested the vectors in vivo in two experimental settings, that is, LLC tumor growth or establishment, in C57BL/6 mice. The treatment of pre-established subcutaneous tumors with the recombinant vectors with ganciclovir (GCV) induced a significant reduction of tumor growth rate, while the in vitro infection of LLC cells with the antiangiogenic vectors before their implantation in mice flanks, either in presence or absence of GCV, completely abolished the tumor establishment. PMID:17557110

  9. Resistance of a human serum-selected human immunodeficiency virus type 1 escape mutant to neutralization by CD4 binding site monoclonal antibodies is conferred by a single amino acid change in gp120.

    PubMed Central

    McKeating, J A; Bennett, J; Zolla-Pazner, S; Schutten, M; Ashelford, S; Brown, A L; Balfe, P

    1993-01-01

    We have selected an HXB2 variant which can replicate in the presence of a neutralizing human serum. Sequencing of the gp120 region of the env gene from the variant and parental viruses identified a single amino acid substitution in the third conserved region of gp120 at residue 375 (AGT-->AAT, Ser-->Asn; designated 375 S/N). The escape mutant was found to be resistant to neutralization by soluble CD4 (sCD4) and four monoclonal antibodies (MAbs), 39.13g, 1.5e, G13, and 448, binding to epitopes overlapping that of the CD4 binding site (CD4 b.s.). Introduction of the 375 S/N mutation into HXB2 by site-directed mutagenesis confirmed that this mutation is responsible for the neutralization-resistant phenotype. Both sCD4 and three of the CD4 b.s. MAbs (39.13g, 1.5e, and G13) demonstrated reduced binding to the native 375 S/N mutant gp120. The ability to select for an escape variant resistant to multiple independent CD4 b.s. MAbs by a human serum confirms the reports that antibodies to the discontinuous CD4 b.s. are a major component of the group-specific neutralizing activity in human sera. PMID:7688820

  10. Cyclic mismatch binding ligand CMBL4 binds to the 5′-T-3′/5′-GG-3′ site by inducing the flipping out of thymine base

    PubMed Central

    Mukherjee, Sanjukta; Dohno, Chikara; Asano, Kaori; Nakatani, Kazuhiko

    2016-01-01

    A newly designed cyclic bis-naphthyridine carbamate dimer CMBL4 with a limited conformational flexibility was synthesized and characterized. Absorption spectra revealed that two naphthyridines in CMBL4 were stacked on each other in aqueous solutions. The most efficient binding of CMBL4 to DNA was observed for the sequence 5′-T-3′/5′-GG-3′ (T/GG) with the formation of a 1:1 complex, which is one of possible structural elements involved in the higher order structures of (TGG)n repeat DNA triggering the genome microdeletion. Surface plasmon resonance assay also showed the binding of CMBL4 with TGG repeat DNA. Potassium permanganate oxidation studies of CMBL4-bound duplex containing the T/GG site showed that the CMBL4-binding accelerated the oxidation of thymine at that site, which suggests the flipping out of the thymine base from a π-stack. Preferential binding was observed for CMBL4 compared with its acyclic variants, which suggests the marked significance of the macrocyclic structure for the recognition of the T/GG site. PMID:27466390

  11. A syndecan-4 binding peptide derived from laminin 5 uses a novel PKCε pathway to induce cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells.

    PubMed

    Filla, Mark S; Clark, Ross; Peters, Donna M

    2014-10-01

    In this study, we examined the role(s) of syndecan-4 in regulating the formation of an actin geodesic dome structure called a cross-linked actin network (CLAN) in which syndecan-4 has previously been localized. CLANs have been described in several different cell types, but they have been most widely studied in human trabecular meshwork (HTM) cells where they may play a key role in controlling intraocular pressure by regulating aqueous humor outflow from the eye. In this study we show that a loss of cell surface synedcan-4 significantly reduces CLAN formation in HTM cells. Analysis of HTM cultures treated with or without dexamethasone shows that laminin 5 deposition within the extracellular matrix is increased by glucocorticoid treatment and that a laminin 5-derived, syndecan-4-binding peptide (PEP75), induces CLAN formation in TM cells. This PEP75-induced CLAN formation was inhibited by heparin and the broad spectrum PKC inhibitor Ro-31-7549. In contrast, the more specific PKCα inhibitor Gö 6976 had no effect, thus excluding PKCα as a downstream effector of syndecan-4 signaling. Analysis of PKC isozyme expression showed that HTM cells also expressed both PKCγ and PKCε. Cells treated with a PKCε agonist formed CLANs while a PKCα/γ agonist had no effect. These data suggest that syndecan-4 is essential for CLAN formation in HTM cells and that a novel PKCε-mediated signaling pathway can regulate formation of this unique actin structure. PMID:25128150

  12. A syndecan-4 binding peptide derived from laminin 5 uses a novel PKCε pathway to induce cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells

    PubMed Central

    Filla, Mark S.; Clark, Ross; Peters, Donna M.

    2014-01-01

    In this study, we examined the role(s) of syndecan-4 in regulating the formation of an actin geodesic dome structure called a cross-linked actin network (CLAN) in which syndecan-4 has previously been localized. CLANs have been described in several different cell types, but they have been most widely studied in human trabecular meshwork (HTM) cells where they may play a key role in controlling intraocular pressure by regulating aqueous humor outflow from the eye. In this study we show that a loss of cell surface synedcan-4 significantly reduces CLAN formation in HTM cells. Analysis of HTM cultures treated with or without dexamethasone shows that laminin 5 deposition within the extracellular matrix is increased by glucocorticoid treatment and that a laminin 5-derived, syndecan-4-binding peptide (PEP75), induces CLAN formation in TM cells. This PEP75-induced CLAN formation was inhibited by heparin and the broad spectrum PKC inhibitor Ro-31-7549. In contrast, the more specific PKCα inhibitor Go 6976 had no effect, thus excluding PKCα as a downstream effector of syndecan-4 signaling. Analysis of PKC isozyme expression showed that HTM cells also expressed both PKCγ and PKCε. Cells treated with a PKCε agonist formed CLANs while a PKCα/γ agonist had no effect. These data suggest that syndecan-4 is essential for CLAN formation in HTM cells and that a novel PKCε-mediated signaling pathway can regulate formation of this unique actin structure. PMID:25128150

  13. Processing, fusogenicity, virion incorporation and CXCR4-binding activity of a feline immunodeficiency virus envelope glycoprotein lacking the two conserved N-glycosylation sites at the C-terminus of the V3 domain.

    PubMed

    González, Silvia A; Affranchino, José L

    2016-07-01

    The process of feline immunodeficiency virus (FIV) entry into its target cells is initiated by the association of the surface (SU) subunit of the viral envelope glycoprotein (Env) with the cellular receptors CD134 and CXCR4. This event is followed by the fusion of the viral and cellular membranes, which is mediated by the transmembrane (TM) subunit of Env. We and others have previously demonstrated that the V3 domain of the SU subunit of Env is essential for CXCR4 binding. Of note, there are two contiguous and highly conserved potential N-glycosylation sites ((418)NST(420) and (422)NLT(424)) located at the C-terminal side of the V3 domain. We therefore decided to study the relevance for Env functions of these N-glycosylation motifs and found that disruption of both of them by introducing the N418Q/N422Q double amino acid substitution drastically impairs Env processing into the SU and TM subunits. Moreover, the simultaneous mutation of these N-glycosylation sites prevents Env incorporation into virions and Env-mediated cell-to-cell fusion. Notably, a recombinant soluble version of the SU glycoprotein carrying the double amino acid replacement N418Q/N422Q at the V3 C-terminal side binds to CXCR4 with an efficiency similar to that of wild-type SU. PMID:27020572

  14. A single amino acid substitution in the C4 region in gp120 confers enhanced neutralization of HIV-1 by modulating CD4 binding sites and V3 loop.

    PubMed

    Ringe, Rajesh; Sharma, Deepak; Zolla-Pazner, Susan; Phogat, Sanjay; Risbud, Arun; Thakar, Madhuri; Paranjape, Ramesh; Bhattacharya, Jayanta

    2011-09-30

    Identification of vulnerability in the HIV-1 envelope (Env) will aid in Env-based vaccine design. We recently found an HIV-1 clade C Env clone (4-2.J45) amplified from a recently infected Indian patient showing exceptional neutralization sensitivity to autologous plasma in contrast to other autologous Envs obtained at the same time point. By constructing chimeric Envs and fine mapping between sensitive and resistant Env clones, we found that substitution of highly conserved isoleucine (I) with methionine (M) (ATA to ATG) at position 424 in the C4 domain conferred enhanced neutralization sensitivity of Env-pseudotyped viruses to autologous and heterologous plasma antibodies. When tested against monoclonal antibodies targeting different sites in gp120 and gp41, Envs expressing M424 showed significant sensitivity to anti-V3 monoclonal antibodies and modestly to sCD4 and b12. Substitution of I424M in unrelated Envs also showed similar neutralization phenotype, indicating that M424 in C4 region induces exposure of neutralizing epitopes particularly in CD4 binding sites and V3 loop. PMID:21851958

  15. Study of antibody repertoires to the CD4 binding site of gp120 of a Chinese HIV-1-infected elite neutralizer, using 454 sequencing and single-cell sorting.

    PubMed

    Li, Dan; Wang, Zheng; Ren, Li; Zhang, Jing; Feng, Guangda; Hong, Kunxue; Hao, Yanling; Qi, Zhi; Liang, Hua; Shao, Yiming

    2016-04-01

    Broadly neutralizing antibodies (NAbs) against the CD4 binding site of HIV gp120 (CD4bs) have provided important information for vaccine design. In this study, we combined deep sequencing and single memory B cell sorting to isolate CD4bs-directed NAbs from a Chinese HIV-1-infected elite neutralizer. We first performed 454 pyrosequencing to capture the IGHV1, IGKV, and IGLV germline gene families. IGHV1-2*02, the heavy chain germline V gene (VH) of the CD4bs-directed bNAb VRC01, was found to have a relatively low somatic mutation rate. When an identity/divergence plot was used to interrogate the 454 sequencing data, no VRC01-like sequences were found within the dataset. We next used a pair of CD4bs-specific probes (RSC3/ΔRSC3) to sort the B cells from this Chinese donor and identified a CD4bs-directed Ab that showed limited neutralization capability. Interestingly, the VH gene of this weak NAb belongs to the IGHV5-51 lineage, with a somatic mutation rate of 7.99 %. Our study thus demonstrates that CD4bs-directed NAbs can be produced by rearrangement from other VH genes, such as IGHV5-51 in this donor, rather than IGHV1-2*02. The 454 sequencing data and NAb obtained from this study will provide useful insights into the CD4bs-directed B-cell response during HIV-1 infection as well as the diversity of neutralizing antibodies. PMID:26671829

  16. Slow formation of stable complexes during coincubation of minimal rRNA and ribosomal protein S4.

    PubMed

    Mayerle, Megan; Bellur, Deepti L; Woodson, Sarah A

    2011-09-23

    Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5' domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation. PMID:21821049

  17. Activation of a human chromosomal replication origin by protein tethering

    PubMed Central

    Chen, Xiaomi; Liu, Guoqi; Leffak, Michael

    2013-01-01

    The specification of mammalian chromosomal replication origins is incompletely understood. To analyze the assembly and activation of prereplicative complexes (pre-RCs), we tested the effects of tethered binding of chromatin acetyltransferases and replication proteins on chromosomal c-myc origin deletion mutants containing a GAL4-binding cassette. GAL4DBD (DNA binding domain) fusions with Orc2, Cdt1, E2F1 or HBO1 coordinated the recruitment of the Mcm7 helicase subunit, the DNA unwinding element (DUE)-binding protein DUE-B and the minichromosome maintenance (MCM) helicase activator Cdc45 to the replicator, and restored origin activity. In contrast, replication protein binding and origin activity were not stimulated by fusion protein binding in the absence of flanking c-myc DNA. Substitution of the GAL4-binding site for the c-myc replicator DUE allowed Orc2 and Mcm7 binding, but eliminated origin activity, indicating that the DUE is essential for pre-RC activation. Additionally, tethering of DUE-B was not sufficient to recruit Cdc45 or activate pre-RCs formed in the absence of a DUE. These results show directly in a chromosomal background that chromatin acetylation, Orc2 or Cdt1 suffice to recruit all downstream replication initiation activities to a prospective origin, and that chromosomal origin activity requires singular DNA sequences. PMID:23658226

  18. Surface plasmon resonance assay of inhibition by pharmaceuticals for thyroxine hormone binging to transport proteins.

    PubMed

    Kinouchi, Hiroki; Matsuyama, Keigo; Kitagawa, Hiroshi; Kamimori, Hiroshi

    2016-01-01

    We developed a surface plasmon resonance (SPR) assay to estimate the competitive inhibition by pharmaceuticals for thyroxine (T4) binding to thyroid hormone transport proteins, transthyretin (TTR) and thyroxine binding globulin (TBG). In this SPR assay, the competitive inhibition of pharmaceuticals for introducing T4 into immobilized TTR or TBG on the sensor chip can be estimated using a running buffer containing pharmaceuticals. The SPR assay showed reproducible immobilization of TTR and TBG, and the kinetic binding parameters of T4 to TTR or TBG were estimated. The equilibrium dissociation constants of TTR or TBG measured by SPR did not clearly differ from data reported for other binding assays. To estimate the competitive inhibition of tetraiodothyroacetic acid, diclofenac, genistein, ibuprofen, carbamazepine, and furosemide, reported to be competitive or noncompetitive pharmaceuticals for T4 binding to TTR or TBG, their 50% inhibition concentrations (IC50) (or 80% inhibition concentration, IC80) were calculated from the change of T4 responses in sensorgrams obtained with various concentrations of the pharmaceuticals. Our SPR method should be a useful tool for predicting the potential of thyroid toxicity of pharmaceuticals by evaluating the competitive inhibition of T4 binding to thyroid hormone binding proteins, TTR and TBG. PMID:26384643

  19. The Structure of an LIM-Only Protein 4 (LMO4) and Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Complex Reveals a Common Mode of Binding to LMO4

    PubMed Central

    Joseph, Soumya; Kwan, Ann H.; Stokes, Philippa H.; Mackay, Joel P.; Cubeddu, Liza; Matthews, Jacqueline M.

    2014-01-01

    LIM-domain only protein 4 (LMO4) is a widely expressed protein with important roles in embryonic development and breast cancer. It has been reported to bind many partners, including the transcription factor Deformed epidermal autoregulatory factor-1 (DEAF1), with which LMO4 shares many biological parallels. We used yeast two-hybrid assays to show that DEAF1 binds both LIM domains of LMO4 and that DEAF1 binds the same face on LMO4 as two other LMO4-binding partners, namely LIM domain binding protein 1 (LDB1) and C-terminal binding protein interacting protein (CtIP/RBBP8). Mutagenic screening analysed by the same method, indicates that the key residues in the interaction lie in LMO4LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4LIM2-DEAF1 complex and determined the solution structure of that complex. Although the LMO4-binding domain from DEAF1 is intrinsically disordered, it becomes structured on binding. The structure confirms that LDB1, CtIP and DEAF1 all bind to the same face on LMO4. LMO4 appears to form a hub in protein-protein interaction networks, linking numerous pathways within cells. Competitive binding for LMO4 therefore most likely provides a level of regulation between those different pathways. PMID:25310299

  20. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  1. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  2. Total protein

    MedlinePlus

    The total protein test measures the total amount of two classes of proteins found in the fluid portion of your ... nutritional problems, kidney disease or liver disease . If total protein is abnormal, you will need to have more ...

  3. Storage Proteins

    PubMed Central

    Fujiwara, Toru; Nambara, Eiji; Yamagishi, Kazutoshi; Goto, Derek B.; Naito, Satoshi

    2002-01-01

    Plants accumulate storage substances such as starch, lipids and proteins in certain phases of development. Storage proteins accumulate in both vegetative and reproductive tissues and serve as a reservoir to be used in later stages of plant development. The accumulation of storage protein is thus beneficial for the survival of plants. Storage proteins are also an important source of dietary plant proteins. Here, we summarize the genome organization and regulation of gene expression of storage protein genes in Arabidopsis. PMID:22303197

  4. Urokinase-type Plasminogen Activator (uPA) Promotes Angiogenesis by Attenuating Proline-rich Homeodomain Protein (PRH) Transcription Factor Activity and De-repressing Vascular Endothelial Growth Factor (VEGF) Receptor Expression.

    PubMed

    Stepanova, Victoria; Jayaraman, Padma-Sheela; Zaitsev, Sergei V; Lebedeva, Tatiana; Bdeir, Khalil; Kershaw, Rachael; Holman, Kelci R; Parfyonova, Yelena V; Semina, Ekaterina V; Beloglazova, Irina B; Tkachuk, Vsevolod A; Cines, Douglas B

    2016-07-15

    Urokinase-type plasminogen activator (uPA) regulates angiogenesis and vascular permeability through proteolytic degradation of extracellular matrix and intracellular signaling initiated upon its binding to uPAR/CD87 and other cell surface receptors. Here, we describe an additional mechanism by which uPA regulates angiogenesis. Ex vivo VEGF-induced vascular sprouting from Matrigel-embedded aortic rings isolated from uPA knock-out (uPA(-/-)) mice was impaired compared with vessels emanating from wild-type mice. Endothelial cells isolated from uPA(-/-) mice show less proliferation and migration in response to VEGF than their wild type counterparts or uPA(-/-) endothelial cells in which expression of wild type uPA had been restored. We reported previously that uPA is transported from cell surface receptors to nuclei through a mechanism that requires its kringle domain. Intranuclear uPA modulates gene transcription by binding to a subset of transcription factors. Here we report that wild type single-chain uPA, but not uPA variants incapable of nuclear transport, increases the expression of cell surface VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) by translocating to the nuclei of ECs. Intranuclear single-chain uPA binds directly to and interferes with the function of the transcription factor hematopoietically expressed homeodomain protein or proline-rich homeodomain protein (HHEX/PRH), which thereby lose their physiologic capacity to repress the activity of vehgr1 and vegfr2 gene promoters. These studies identify uPA-dependent de-repression of vegfr1 and vegfr2 gene transcription through binding to HHEX/PRH as a novel mechanism by which uPA mediates the pro-angiogenic effects of VEGF and identifies a potential new target for control of pathologic angiogenesis. PMID:27151212

  5. Dietary Proteins

    MedlinePlus

    ... grains and beans. Proteins from meat and other animal products are complete proteins. This means they supply all of the amino acids the body can't make on its own. Most plant proteins are incomplete. You should eat different types of plant proteins every day to get ...

  6. Protein Analysis

    NASA Astrophysics Data System (ADS)

    Chang, Sam K. C.

    Proteins are an abundant component in all cells, and almost all except storage proteins are important for biological functions and cell structure. Food proteins are very complex. Many have been purified and characterized. Proteins vary in molecular mass, ranging from approximately 5000 to more than a million Daltons. They are composed of elements including hydrogen, carbon, nitrogen, oxygen, and sulfur. Twenty α-amino acids are the building blocks of proteins; the amino acid residues in a protein are linked by peptide bonds. Nitrogen is the most distinguishing element present in proteins. However, nitrogen content in various food proteins ranges from 13.4 to 19.1% (1) due to the variation in the specific amino acid composition of proteins. Generally, proteins rich in basic amino acids contain more nitrogen.

  7. Total protein

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  8. Whey Protein

    MedlinePlus

    ... shows that taking whey protein in combination with strength training increases lean body mass, strength, and muscle size. ... grams/kg of whey protein in combination with strength training for 6-10 weeks. For HIV/AIDS-related ...

  9. Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ricard-Blum, S.

    Proteins are key actors in the life of the cell, involved in many physiological and pathological processes. Since variations in the expression of messenger RNA are not systematically correlated with variations in the protein levels, the latter better reflect the way a cell functions. Protein microarrays thus supply complementary information to DNA chips. They are used in particular to analyse protein expression profiles, to detect proteins within complex biological media, and to study protein-protein interactions, which give information about the functions of those proteins [3-9]. They have the same advantages as DNA microarrays for high-throughput analysis, miniaturisation, and the possibility of automation. Section 18.1 gives a brief overview of proteins. Following this, Sect. 18.2 describes how protein microarrays can be made on flat supports, explaining how proteins can be produced and immobilised on a solid support, and discussing the different kinds of substrate and detection method. Section 18.3 discusses the particular format of protein microarrays in suspension. The diversity of protein microarrays and their applications are then reported in Sect. 18.4, with applications to therapeutics (protein-drug interactions) and diagnostics. The prospects for future developments of protein microarrays are then outlined in the conclusion. The bibliography provides an extensive list of reviews and detailed references for those readers who wish to go further in this area. Indeed, the aim of the present chapter is not to give an exhaustive or detailed analysis of the state of the art, but rather to provide the reader with the basic elements needed to understand how proteins are designed and used.

  10. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  11. Identification of multiple proteins expressed in murine embryos as binding partners for the WW domains of the ubiquitin-protein ligase Nedd4.

    PubMed Central

    Jolliffe, C N; Harvey, K F; Haines, B P; Parasivam, G; Kumar, S

    2000-01-01

    Nedd4 is a member of a growing family of ubiquitin-protein ligases which consist of a lipid-binding domain, two to four WW domains and a C-terminal ubiquitin-protein ligase domain. The Nedd4 mRNA levels are developmentally regulated and Nedd4 protein is highly expressed in many mouse embryonic tissues. In this study we have used a far-Western screen to identify embryonic proteins that interact with the WW domains in mouse Nedd4. We report here identification of eight Nedd4 WW-domain-interacting proteins from mouse embryonic cDNA expression libraries. Two of the proteins are novel, while two have been identified previously as ligands for a WW domain. All of these proteins contain one or more PY motifs. In seven of the eight proteins, these PY motifs are necessary for their interaction with the WW domains of Nedd4. Using site-directed mutagenesis, and by using individual WW domains of Nedd4 as probes for far-Western analysis, we show that the three WW domains in Nedd4 interact with varying affinities with the PY motifs present in various Nedd4-binding proteins. These results provide evidence that Nedd4 can potentially interact with multiple proteins, possibly simultaneously, through its WW domains. PMID:11042109

  12. Specific interactions of the L10(L12)4 ribosomal protein complex with mRNA, rRNA, and L11.

    PubMed

    Iben, James R; Draper, David E

    2008-03-01

    Large ribosomal subunit proteins L10 and L12 form a pentameric protein complex, L10(L12) 4, that is intimately involved in the ribosome elongation cycle. Its contacts with rRNA or other ribosomal proteins have been only partially resolved by crystallography. In Escherichia coli, L10 and L12 are encoded from a single operon for which L10(L12) 4 is a translational repressor that recognizes a secondary structure in the mRNA leader. In this study, L10(L12) 4 was expressed from the moderate thermophile Bacillus stearothermophilus to quantitatively compare strategies for binding of the complex to mRNA and ribosome targets. The minimal mRNA recognition structure is widely distributed among bacteria and has the potential to form a kink-turn structure similar to one identified in the rRNA as part of the L10(L12) 4 binding site. Mutations in equivalent positions between the two sequences have similar effects on L10(L12) 4-RNA binding affinity and identify the kink-turn motif and a loop AA sequence as important recognition elements. In contrast to the larger rRNA structure, the mRNA apparently positions the kink-turn motif and loop for protein recognition without the benefit of Mg (2+)-dependent tertiary structure. The mRNA and rRNA fragments bind L10(L12) 4 with similar affinity ( approximately 10 (8) M (-1)), but fluorescence binding studies show that a nearby protein in the ribosome, L11, enhances L10(L12) 4 binding approximately 100-fold. Thus, mRNA and ribosome targets use similar RNA features, held in different structural contexts, to recognize L10(L12) 4, and the ribosome ensures the saturation of its L10(L12) 4 binding site by means of an additional protein-protein interaction. PMID:18247578

  13. Protein folds and protein folding

    PubMed Central

    Schaeffer, R. Dustin; Daggett, Valerie

    2011-01-01

    The classification of protein folds is necessarily based on the structural elements that distinguish domains. Classification of protein domains consists of two problems: the partition of structures into domains and the classification of domains into sets of similar structures (or folds). Although similar topologies may arise by convergent evolution, the similarity of their respective folding pathways is unknown. The discovery and the characterization of the majority of protein folds will be followed by a similar enumeration of available protein folding pathways. Consequently, understanding the intricacies of structural domains is necessary to understanding their collective folding pathways. We review the current state of the art in the field of protein domain classification and discuss methods for the systematic and comprehensive study of protein folding across protein fold space via atomistic molecular dynamics simulation. Finally, we discuss our large-scale Dynameomics project, which includes simulations of representatives of all autonomous protein folds. PMID:21051320

  14. Protein Dynamics

    NASA Astrophysics Data System (ADS)

    Frauenfelder, Hans

    2011-03-01

    Proteins combine properties of solids, liquids, and glasses. Schrödinger anticipated the main features of biomolecules long ago by stating that they had to be solid-like, but able to assume many different conformations. Indeed proteins can assume a gigantic number of conformational substates with the same primary sequence but different conformations. The different substates are described as craters in a very-high-dimensional energy landscape. The energy landscape is organized in a hierarchy of tiers, craters within craters within craters. Protein motions are pictured as transition between substates - jumps from crater to crater. Initially we assumed that these jumps were controlled by internal barriers between substates, but experiments have shown that nature selected a different approach. Proteins are surrounded by one to two layers of water and are embedded in a bulk solvent. Structural motions of the protein are controlled by the alpha fluctuations in the solvent surrounding the protein. Some internal motions most likely involving side chains are controlled electrostatically by beta fluctuations in the hydration shell. The dynamics of proteins is consequently dominated by the environment (H. Frauenfelder et al. PNAS 106, 5129 (2009). One can speculate that this organization permits exchange of information among biomolecules. The energy landscape is not just organized into two tiers, alpha and beta, but cryogenic experiments have revealed more tiers and protein more properties similar to that of glasses. While proteins function at ambient temperatures, cryogenic studies are necessary to understand the physics relevant for biology.

  15. Interfacial Protein-Protein Associations

    PubMed Central

    Langdon, Blake B.; Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.

    2014-01-01

    While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on poly(ethylene glycol) modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for longer times. The appearance of three distinct RET states suggested a spatially heterogeneous surface – with areas of high protein density (i.e. strongly-interacting clusters) coexisting with mobile monomers. Distinct association states exhibited characteristic behavior, i.e. partial-RET (monomer-monomer) associations were shorter-lived than complete-RET (protein-cluster) associations. While the fractional surface area covered by regions with high protein density (i.e. clusters) increased with increasing concentration, the distribution of contact times between monomers and clusters was independent of solution concentration, suggesting that associations were a local phenomenon, and independent of the global surface coverage. PMID:24274729

  16. Magnetic circular dichroism spectroscopic characterization of the NOS-like protein from Geobacillus stearothermophilus (gsNOS).

    PubMed

    Kinloch, Ryan D; Sono, Masanori; Sudhamsu, Jawahar; Crane, Brian R; Dawson, John H

    2010-03-01

    Nitric oxide synthase (NOS) catalyzes the NADPH- and O(2)-dependent oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline via an N(G)-hydroxy-l-arginine (NHA) intermediate. Mammalian NOSs have been studied quite extensively; other eukaryotes and some prokaryotes appear to express NOS-like proteins comparable to the oxygenase domain of mammalian NOSs. In this study, a recombinant NOS-like protein from the thermostable bacterium Geobacillus stearothermophilus (gsNOS) has been characterized using magnetic circular dichroism (MCD) and UV-Vis absorption spectroscopic techniques. Spectral comparisons of ligand complexes (with O(2), NO and CO) of substrate-bound (l-Arg or NHA) gsNOS, including the key oxyferrous complex studied at -50 degrees C in cryogenic mixed solvents, with analogous mammalian NOS complexes indicate overall spectroscopic similarities between gsNOS and mammalian NOSs. However, more detailed spectral comparisons reflect subtle structural differences between gsNOS and mammalian NOSs. This may be due to an incomplete tetrahydrobiopterin (BH(4))-binding site and low BH(4)-binding affinity, which may become even lower in the presence of cryosolvent in gsNOS. Although BH(4)-binding may be altered, gsNOS appears to require the pterin for NO production since formation of the stable ferric-NO product complex was only observed when excess BH(4) (>150muM) over gsNOS was present upon single turnover reaction in which O(2) was bubbled into dithionite-reduced NHA-bound protein solution at -35 degrees C or -50 degrees C. PMID:20110129

  17. Invertebrate and Vertebrate Class III Myosins Interact with MORN Repeat-Containing Adaptor Proteins

    PubMed Central

    Mecklenburg, Kirk L.; Freed, Stephanie A.; Raval, Manmeet; Quintero, Omar A.; Yengo, Christopher M.; O'Tousa, Joseph. E.

    2015-01-01

    In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP). Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A). In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior. PMID:25822849

  18. Whey Protein

    MedlinePlus

    ... intolerance, for replacing or supplementing milk-based infant formulas, and for reversing weight loss and increasing glutathione ( ... allergic reactions compared to infants who receive standard formula. However, taking why protein might not be helpful ...

  19. Designed protein-protein association.

    PubMed

    Grueninger, Dirk; Treiber, Nora; Ziegler, Mathias O P; Koetter, Jochen W A; Schulze, Monika-Sarah; Schulz, Georg E

    2008-01-11

    The analysis of natural contact interfaces between protein subunits and between proteins has disclosed some general rules governing their association. We have applied these rules to produce a number of novel assemblies, demonstrating that a given protein can be engineered to form contacts at various points of its surface. Symmetry plays an important role because it defines the multiplicity of a designed contact and therefore the number of required mutations. Some of the proteins needed only a single side-chain alteration in order to associate to a higher-order complex. The mobility of the buried side chains has to be taken into account. Four assemblies have been structurally elucidated. Comparisons between the designed contacts and the results will provide useful guidelines for the development of future architectures. PMID:18187656

  20. Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets.

    PubMed

    Nagy, Zoltan; Wynne, Kieran; von Kriegsheim, Alexander; Gambaryan, Stepan; Smolenski, Albert

    2015-12-11

    Endothelial cells release prostacyclin (PGI2) and nitric oxide (NO) to inhibit platelet functions. PGI2 and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition. PMID:26507661

  1. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  2. Human C3b- and C4b-regulatory proteins: a new multi-gene family.

    PubMed

    Holers, V M; Cole, J L; Lublin, D M; Seya, T; Atkinson, J P

    1985-06-01

    The complement cascade is regulated to prevent inappropriate activation. This regulation is targeted not only at the initiation of the cascade but also at the amplification andjunctional (C3) steps. Five glycoproteins with both complement regulatory activity and binding affinity for C3b/C4b have been characterized. In plasma these molecules are factor H(H) and C4-binding protein (C4-bp) and, on cells, they are the C3b/C4b receptor (CR1), decay-accelerating factor (DAF), and gp45-70. Here Michael Holers and his colleagues review structural, functional and genetic studies of these proteins and discuss the evidence for a new multi-gene family with a common ancestral protein. PMID:25289982

  3. Bacteriophage protein-protein interactions.

    PubMed

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian; Uetz, Peter

    2012-01-01

    Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  4. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  5. Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes.

    PubMed

    Yoneyama, T; Hatakeyama, K

    2001-04-01

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed-forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 microM, and phenylalanine binds to the protein complex with a Kd of 94 microM. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively. PMID:11274478

  6. Liver Retinol Transporter and Receptor for Serum Retinol-binding Protein (RBP4)*

    PubMed Central

    Alapatt, Philomena; Guo, Fangjian; Komanetsky, Susan M.; Wang, Shuping; Cai, Jinjin; Sargsyan, Ashot; Rodríguez Díaz, Eduardo; Bacon, Brandon T.; Aryal, Pratik; Graham, Timothy E.

    2013-01-01

    Vitamin A (retinol) is absorbed in the small intestine, stored in liver, and secreted into circulation bound to serum retinol-binding protein (RBP4). Circulating retinol may be taken up by extrahepatic tissues or recycled back to liver multiple times before it is finally metabolized or degraded. Liver exhibits high affinity binding sites for RBP4, but specific receptors have not been identified. The only known high affinity receptor for RBP4, Stra6, is not expressed in the liver. Here we report discovery of RBP4 receptor-2 (RBPR2), a novel retinol transporter expressed primarily in liver and intestine and induced in adipose tissue of obese mice. RBPR2 is structurally related to Stra6 and highly conserved in vertebrates, including humans. Expression of RBPR2 in cultured cells confers high affinity RBP4 binding and retinol transport, and RBPR2 knockdown reduces RBP4 binding/retinol transport. RBPR2 expression is suppressed by retinol and retinoic acid and correlates inversely with liver retinol stores in vivo. We conclude that RBPR2 is a novel retinol transporter that potentially regulates retinol homeostasis in liver and other tissues. In addition, expression of RBPR2 in liver and fat suggests a possible role in mediating established metabolic actions of RBP4 in those tissues. PMID:23105095

  7. Hypothesis: Paralog Formation from Progenitor Proteins and Paralog Mutagenesis Spur the Rapid Evolution of Telomere Binding Proteins.

    PubMed

    Lustig, Arthur J

    2016-01-01

    Through elegant studies in fungal cells and complex organisms, we propose a unifying paradigm for the rapid evolution of telomere binding proteins (TBPs) that associate with either (or both) telomeric DNA and telomeric proteins. TBPs protect and regulate telomere structure and function. Four critical factors are involved. First, TBPs that commonly bind to telomeric DNA include the c-Myb binding proteins, OB-fold single-stranded binding proteins, and G-G base paired Hoogsteen structure (G4) binding proteins. Each contributes independently or, in some cases, cooperatively, to provide a minimum level of telomere function. As a result of these minimal requirements and the great abundance of homologs of these motifs in the proteome, DNA telomere-binding activity may be generated more easily than expected. Second, telomere dysfunction gives rise to genome instability, through the elevation of recombination rates, genome ploidy, and the frequency of gene mutations. The formation of paralogs that diverge from their progenitor proteins ultimately can form a high frequency of altered TBPs with altered functions. Third, TBPs that assemble into complexes (e.g., mammalian shelterin) derive benefits from the novel emergent functions. Fourth, a limiting factor in the evolution of TBP complexes is the formation of mutually compatible interaction surfaces amongst the TBPs. These factors may have different degrees of importance in the evolution of different phyla, illustrated by the apparently simpler telomeres in complex plants. Selective pressures that can utilize the mechanisms of paralog formation and mutagenesis to drive TBP evolution along routes dependent on the requisite physiologic changes. PMID:26904098

  8. Hypothesis: Paralog Formation from Progenitor Proteins and Paralog Mutagenesis Spur the Rapid Evolution of Telomere Binding Proteins

    PubMed Central

    Lustig, Arthur J.

    2016-01-01

    Through elegant studies in fungal cells and complex organisms, we propose a unifying paradigm for the rapid evolution of telomere binding proteins (TBPs) that associate with either (or both) telomeric DNA and telomeric proteins. TBPs protect and regulate telomere structure and function. Four critical factors are involved. First, TBPs that commonly bind to telomeric DNA include the c-Myb binding proteins, OB-fold single-stranded binding proteins, and G-G base paired Hoogsteen structure (G4) binding proteins. Each contributes independently or, in some cases, cooperatively, to provide a minimum level of telomere function. As a result of these minimal requirements and the great abundance of homologs of these motifs in the proteome, DNA telomere-binding activity may be generated more easily than expected. Second, telomere dysfunction gives rise to genome instability, through the elevation of recombination rates, genome ploidy, and the frequency of gene mutations. The formation of paralogs that diverge from their progenitor proteins ultimately can form a high frequency of altered TBPs with altered functions. Third, TBPs that assemble into complexes (e.g., mammalian shelterin) derive benefits from the novel emergent functions. Fourth, a limiting factor in the evolution of TBP complexes is the formation of mutually compatible interaction surfaces amongst the TBPs. These factors may have different degrees of importance in the evolution of different phyla, illustrated by the apparently simpler telomeres in complex plants. Selective pressures that can utilize the mechanisms of paralog formation and mutagenesis to drive TBP evolution along routes dependent on the requisite physiologic changes. PMID:26904098

  9. Protein inference: A protein quantification perspective.

    PubMed

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/. PMID:26935399

  10. Structural basis of protein phosphatase 2A stable latency

    PubMed Central

    Jiang, Li; Stanevich, Vitali; Satyshur, Kenneth A; Kong, Mei; Watkins, Guy R.; Wadzinski, Brian E.; Sengupta, Rituparna; Xing, Yongna

    2013-01-01

    The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay. These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site. The PP2Ac–α4 interface is essential for cell survival and sterically hinders a PP2A ubiquitination site, important for the stability of cellular PP2Ac. Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes. PMID:23591866

  11. Interfacing protein lysine acetylation and protein phosphorylation

    PubMed Central

    Tran, Hue T.; Uhrig, R. Glen; Nimick, Mhairi; Moorhead, Greg B.

    2012-01-01

    Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature. PMID:22827947

  12. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  13. A sterol binding protein integrates endosomal lipid metabolism with TOR signaling and nitrogen sensing

    PubMed Central

    Mousley, Carl J.; Yuan, Peihua; Gaur, Naseem A.; Trettin, Kyle D.; Nile, Aaron H.; Deminoff, Stephen J.; Dewar, Brian J.; Wolpert, Max; Macdonald, Jeffrey M.; Herman, Paul K.; Hinnebusch, Alan G.; Bankaitis, Vytas A.

    2012-01-01

    SUMMARY Kes1, and other oxysterol binding protein (OSBP) superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids, and dampens gene expression driven by Gcn4; the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic ‘large Mediator’ complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing. PMID:22341443

  14. Fibronectin-Binding Protein of Borrelia hermsii Expressed in the Blood of Mice with Relapsing Fever

    PubMed Central

    Lewis, Eric R. G.; Marcsisin, Renee A.; Campeau Miller, Shelley A.; Hue, Fong; Phillips, April; AuCoin, David P.

    2014-01-01

    To identify and characterize surface proteins expressed by the relapsing fever (RF) agent Borrelia hermsii in the blood of infected mice, we used a cell-free filtrate of their blood to immunize congenic naive mice. The resultant antiserum was used for Western blotting of cell lysates, and gel slices corresponding to reactive bands were subjected to liquid chromatography-tandem mass spectrometry, followed by a search of the proteome database with the peptides. One of the immunogens was identified as the BHA007 protein, which is encoded by a 174-kb linear plasmid. BHA007 had sequence features of lipoproteins, was surface exposed by the criteria of in situ protease susceptibility and agglutination of Vtp− cells by anti-BHA007 antibodies, and was not essential for in vitro growth. BHA007 elicited antibodies during experimental infection of mice, but immunization with recombinant protein did not confer protection against needle-delivered infection. Open reading frames (ORFs) orthologous to BHA007 were found on large plasmids of other RF species, including the coding sequences for the CihC proteins of Borrelia duttonii and B. recurrentis, but not in Lyme disease Borrelia species. Recombinant BHA007 bound both human and bovine fibronectin with Kd (dissociation constant) values of 22 and 33 nM, respectively, and bound to C4-binding protein with less affinity. The distant homology of BHA007 and its orthologs to BBK32 proteins of Lyme disease species, as well as to previously described BBK32-like proteins in relapsing fever species, indicates that BHA007 is a member of a large family of multifunctional proteins in Borrelia species that bind to fibronectin as well as other host proteins. PMID:24686059

  15. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  16. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  17. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  18. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  19. Protein folding, protein homeostasis, and cancer

    PubMed Central

    Van Drie, John H.

    2011-01-01

    Proteins fold into their functional 3-dimensional structures from a linear amino acid sequence. In vitro this process is spontaneous; while in vivo it is orchestrated by a specialized set of proteins, called chaperones. Protein folding is an ongoing cellular process, as cellular proteins constantly undergo synthesis and degradation. Here emerging links between this process and cancer are reviewed. This perspective both yields insights into the current struggle to develop novel cancer chemotherapeutics and has implications for future chemotherapy discovery. PMID:21272445

  20. Split-Protein Systems: Beyond Binary Protein-Protein Interactions

    PubMed Central

    Shekhawat, Sujan S.; Ghosh, Indraneel

    2011-01-01

    It has been estimated that 650,000 protein-protein interactions exist in the human interactome [1], a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, E. coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. PMID:22070901

  1. Split-protein systems: beyond binary protein-protein interactions.

    PubMed

    Shekhawat, Sujan S; Ghosh, Indraneel

    2011-12-01

    It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. PMID:22070901

  2. Protein-losing enteropathy

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  3. Protein electrophoresis - serum

    MedlinePlus

    ... digestive tract to absorb proteins ( protein-losing enteropathy ) Malnutrition Kidney disorder called nephrotic syndrome Scarring of the ... may indicate: Abnormally low level of LDL cholesterol Malnutrition Increased gamma globulin proteins may indicate: Bone marrow ...

  4. Protein in diet

    MedlinePlus

    ... basic structure of protein is a chain of amino acids. You need protein in your diet to help ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a number ...

  5. Leukotriene BLT2 Receptor Monomers Activate the Gi2 GTP-binding Protein More Efficiently than Dimers*

    PubMed Central

    Arcemisbéhère, Laure; Sen, Tuhinadri; Boudier, Laure; Balestre, Marie-Noëlle; Gaibelet, Gérald; Detouillon, Emilie; Orcel, Hélène; Mendre, Christiane; Rahmeh, Rita; Granier, Sébastien; Vivès, Corinne; Fieschi, Franck; Damian, Marjorie; Durroux, Thierry; Banères, Jean-Louis; Mouillac, Bernard

    2010-01-01

    Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B4 receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory Gi2 protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human α5 integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB4 binding in the presence of the purified G protein Gαi2. The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5′-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified Gαi2β1γ2 protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal. PMID:20026606

  6. Drugging Membrane Protein Interactions.

    PubMed

    Yin, Hang; Flynn, Aaron D

    2016-07-11

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind cells to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally "undruggable" regions of membrane proteins, enabling modulation of protein-protein, protein-lipid, and protein-nucleic acid interactions. In this review, we survey the state of the art of high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  7. Domains mediate protein-protein interactions and nucleate protein assemblies.

    PubMed

    Costa, S; Cesareni, G

    2008-01-01

    Cell physiology is governed by an intricate mesh of physical and functional links among proteins, nucleic acids and other metabolites. The recent information flood coming from large-scale genomic and proteomic approaches allows us to foresee the possibility of compiling an exhaustive list of the molecules present within a cell, enriched with quantitative information on concentration and cellular localization. Moreover, several high-throughput experimental and computational techniques have been devised to map all the protein interactions occurring in a living cell. So far, such maps have been drawn as graphs where nodes represent proteins and edges represent interactions. However, this representation does not take into account the intrinsically modular nature of proteins and thus fails in providing an effective description of the determinants of binding. Since proteins are composed of domains that often confer on proteins their binding capabilities, a more informative description of the interaction network would detail, for each pair of interacting proteins in the network, which domains mediate the binding. Understanding how protein domains combine to mediate protein interactions would allow one to add important features to the protein interaction network, making it possible to discriminate between simultaneously occurring and mutually exclusive interactions. This objective can be achieved by experimentally characterizing domain recognition specificity or by analyzing the frequency of co-occurring domains in proteins that do interact. Such approaches allow gaining insights on the topology of complexes with unknown three-dimensional structure, thus opening the prospect of adopting a more rational strategy in developing drugs designed to selectively target specific protein interactions. PMID:18491061

  8. Protein sensing with engineered protein nanopores*

    PubMed Central

    Mohammad, Mohammad M.; Movileanu, Liviu

    2013-01-01

    The use of nanopores is a powerful new frontier in single-molecule sciences. Nanopores have been used effectively in exploring various biophysical features of small polypeptides and proteins, such as their folding state and structure, ligand interactions, and enzymatic activity. In particular, the α-hemolysin protein pore (αHL) has been used extensively for the detection, characterization and analysis of polypeptides, because this protein nanopore is highly robust, versatile and tractable under various experimental conditions. Inspired by the mechanisms of protein translocation across the outer membrane translocases of mitochondria, we have shown the ability to use nanopore-probe techniques in controlling a single protein using engineered αHL pores. Here, we provide a detailed protocol for the preparation of αHL protein nanopores. Moreover, we demonstrate that placing attractive electrostatic traps is instrumental in tackling single-molecule stochastic sensing of folded proteins. PMID:22528256

  9. Nanotechnologies in protein microarrays.

    PubMed

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures. PMID:26039143

  10. Targeting prion-like protein doppel selectively suppresses tumor angiogenesis

    PubMed Central

    Al-Hilal, Taslim A.; Chung, Seung Woo; Choi, Jeong Uk; Kim, Seong Who; Kim, Sang Yoon; Ahsan, Fakhrul; Kim, In-San

    2016-01-01

    Controlled and site-specific regulation of growth factor signaling remains a major challenge for current antiangiogenic therapies, as these antiangiogenic agents target normal vasculature as well tumor vasculature. In this article, we identified the prion-like protein doppel as a potential therapeutic target for tumor angiogenesis. We investigated the interactions between doppel and VEGFR2 and evaluated whether blocking the doppel/VEGFR2 axis suppresses the process of angiogenesis. We discovered that tumor endothelial cells (TECs), but not normal ECs, express doppel; tumors from patients and mouse xenografts expressed doppel in their vasculatures. Induced doppel overexpression in ECs enhanced vascularization, whereas doppel constitutively colocalized and complexed with VEGFR2 in TECs. Doppel inhibition depleted VEGFR2 from the cell membrane, subsequently inducing the internalization and degradation of VEGFR2 and thereby attenuating VEGFR2 signaling. We also synthesized an orally active glycosaminoglycan (LHbisD4) that specifically binds with doppel. We determined that LHbisD4 concentrates over the tumor site and that genetic loss of doppel in TECs decreases LHbisD4 binding and targeting both in vitro and in vivo. Moreover, LHbisD4 eliminated VEGFR2 from the cell membrane, prevented VEGF binding in TECs, and suppressed tumor growth. Together, our results demonstrate that blocking doppel can control VEGF signaling in TECs and selectively inhibit tumor angiogenesis. PMID:26950422

  11. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  12. Protein sequence comparison and protein evolution

    SciTech Connect

    Pearson, W.R.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  13. Whey protein fractionation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concentrated whey protein products from cheese whey, such as whey protein concentrate (WPC) and whey protein isolate (WPI), contain more than seven different types of proteins: alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), bovine serum albumin (BSA), immunoglobulins (Igs), lactoferrin ...

  14. Sorghum and millet proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum and millet proteins are an important source of dietary protein for significant numbers of people living throughout Africa and parts of Asia. Compared to other food proteins, such as those found in milk, eggs and wheat, little is known about the functionality of sorghum and millet proteins. ...

  15. Protein in diet

    MedlinePlus

    ... protein. The basic structure of protein is a chain of amino acids. You need protein in your diet to help your body repair cells and make new ones. Protein is also important for growth and development in children, teens, and pregnant women.

  16. Techniques in protein methylation.

    PubMed

    Lee, Jaeho; Cheng, Donghang; Bedford, Mark T

    2004-01-01

    Proteins can be methylated on the side-chain nitrogens of arginine and lysine residues or on carboxy-termini. Protein methylation is a way of subtly changing the primary sequence of a peptide so that it can encode more information. This common posttranslational modification is implicated in the regulation of a variety of processes including protein trafficking, transcription and protein-protein interactions. In this chapter, we will use the arginine methyltransferases to illustrate different approaches that have been developed to assess protein methylation. Both in vivo and in vitro methylation techniques are described, and the use of small molecule inhibitors of protein methylation will be demonstrated. PMID:15173617

  17. Urine Protein and Urine Protein to Creatinine Ratio

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Urine Protein and Urine Protein to Creatinine Ratio Share this page: Was this page helpful? Also known as: 24-Hour Urine Protein; Urine Total Protein; Urine Protein to Creatinine Ratio; ...

  18. Biochemical Approaches for Discovering Protein-Protein Interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein-protein interactions or protein complexes are indigenous to nearly all cellular processes, ranging from metabolism to structure. Elucidating both individual protein associations and complex protein interaction networks, while challenging, is an essential goal of functional genomics. For ex...

  19. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  20. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  1. Bromodomain protein 4 mediates the papillomavirus E2 transcriptional activation function.

    PubMed

    Schweiger, Michal-Ruth; You, Jianxin; Howley, Peter M

    2006-05-01

    The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis. PMID:16611886

  2. CD4-Specific Designed Ankyrin Repeat Proteins Are Novel Potent HIV Entry Inhibitors with Unique Characteristics

    PubMed Central

    Schweizer, Andreas; Rusert, Peter; Berlinger, Livia; Ruprecht, Claudia R.; Mann, Axel; Corthésy, Stéphanie; Turville, Stuart G.; Aravantinou, Meropi; Fischer, Marek; Robbiani, Melissa; Amstutz, Patrick; Trkola, Alexandra

    2008-01-01

    Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins—high physical stability, specificity and low production costs—with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development. PMID:18654624

  3. Designing Fluorinated Proteins.

    PubMed

    Marsh, E N G

    2016-01-01

    As methods to incorporate noncanonical amino acid residues into proteins have become more powerful, interest in their use to modify the physical and biological properties of proteins and enzymes has increased. This chapter discusses the use of highly fluorinated analogs of hydrophobic amino acids, for example, hexafluoroleucine, in protein design. In particular, fluorinated residues have proven to be generally effective in increasing the thermodynamic stability of proteins. The chapter provides an overview of the different fluorinated amino acids that have been used in protein design and the various methods available for producing fluorinated proteins. It discusses model proteins systems into which highly fluorinated amino acids have been introduced and the reasons why fluorinated residues are generally stabilizing, with particular reference to thermodynamic and structural studies from our laboratory. Lastly, details of the methodology we have developed to measure the thermodynamic stability of oligomeric fluorinated proteins are presented, as this may be generally applicable to many proteins. PMID:27586337

  4. Surface Mediated Protein Disaggregation

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Kumar, Sanat K.

    2014-03-01

    Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

  5. PINT: Protein-protein Interactions Thermodynamic Database.

    PubMed

    Kumar, M D Shaji; Gromiha, M Michael

    2006-01-01

    The first release of Protein-protein Interactions Thermodynamic Database (PINT) contains >1500 data of several thermodynamic parameters along with sequence and structural information, experimental conditions and literature information. Each entry contains numerical data for the free energy change, dissociation constant, association constant, enthalpy change, heat capacity change and so on of the interacting proteins upon binding, which are important for understanding the mechanism of protein-protein interactions. PINT also includes the name and source of the proteins involved in binding, their Protein Information Resource, SWISS-PROT and Protein Data Bank (PDB) codes, secondary structure and solvent accessibility of residues at mutant positions, measuring methods, experimental conditions, such as buffers, ions and additives, and literature information. A WWW interface facilitates users to search data based on various conditions, feasibility to select the terms for output and different sorting options. Further, PINT is cross-linked with other related databases, PIR, SWISS-PROT, PDB and NCBI PUBMED literature database. The database is freely available at http://www.bioinfodatabase.com/pint/index.html. PMID:16381844

  6. Physical and functional interactions between herpes simplex virus immediate-early proteins ICP4 and ICP27.

    PubMed Central

    Panagiotidis, C A; Lium, E K; Silverstein, S J

    1997-01-01

    The ordered expression of herpes simplex virus type 1 (HSV-1) genes, during the course of a productive infection, requires the action of the virus immediate-early regulatory proteins. Using a protein interaction assay, we demonstrate specific in vitro protein-protein interactions between ICP4 and ICP27, two immediate-early proteins of HSV-1 that are essential for virus replication. We map multiple points of contact between these proteins. Furthermore, by coimmunoprecipitation experiments, we demonstrate the following. (i) ICP4-ICP27 complexes are present in extracts from HSV-1 infected cells. (ii) ICP27 binds preferentially to less modified forms of ICP4, a protein that is extensively modified posttranslationally. We also demonstrate, by performing electrophoretic mobility shift assays and supershifts with monoclonal antibodies to ICP4 or ICP27, that both proteins are present in a DNA-protein complex with a noncanonical ICP4 binding site present in the HSV thymidine kinase (TK) gene. ICP4, in extracts from cells infected with ICP27-deficient viruses, is impaired in its ability to form complexes with the TK site but not with the canonical site from the alpha4 gene. However, ICP4 is able to form complexes with the TK probe, in the absence of ICP27, when overproduced in mammalian cells or expressed in bacteria. These data suggest that the inability of ICP4 from infected cell extracts to bind the TK probe in the absence of ICP27 does not reflect a requirement for the physical presence of ICP27 in the complex. Rather, they imply that ICP27 is likely to modulate the DNA binding activity of ICP4 by affecting its posttranslational modification status. Therefore, we propose that ICP27, in addition to its established role as a posttranscriptional regulator of virus gene expression, may also modulate transcription either through direct or indirect interactions with HSV regulatory regions, or through its ability to modulate the DNA binding activity of ICP4. PMID:8995681

  7. DNA mimicry by proteins.

    PubMed

    Dryden, D T F; Tock, M R

    2006-04-01

    It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins. PMID:16545103

  8. Physics of protein motility and motor proteins

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  9. An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4

    PubMed Central

    Fehler, Olesja; Singh, Priyanka; Haas, Astrid; Ulrich, Diana; Müller, Jan P.; Ohnheiser, Johanna; Klempnauer, Karl-Heinz

    2014-01-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5′-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4. PMID:25190455

  10. An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4.

    PubMed

    Fehler, Olesja; Singh, Priyanka; Haas, Astrid; Ulrich, Diana; Müller, Jan P; Ohnheiser, Johanna; Klempnauer, Karl-Heinz

    2014-01-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5'-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4. PMID:25190455

  11. Protein C blood test

    MedlinePlus

    ... a normal substance in the body that prevents blood clotting. A blood test can be done to see ... history of blood clots. Protein C helps control blood clotting. A lack of this protein or problem with ...

  12. Protein S blood test

    MedlinePlus

    ... a normal substance in your body that prevents blood clotting. A blood test can be done to see ... family history of blood clots. Protein S helps control blood clotting. A lack of this protein or problem with ...

  13. Protein electrophoresis - urine

    MedlinePlus

    ... nephropathy Kidney failure Multiple myeloma Nephrotic syndrome Acute urinary tract infection Risks There are no risks associated with this ... Primary amyloidosis Protein in diet Protein urine test Urinary tract infection - adults Update Date 5/29/2014 Updated by: ...

  14. [Protein-losing enteropathy].

    PubMed

    Amiot, A

    2015-07-01

    Protein-losing enteropathy is a rare syndrome of gastrointestinal protein loss. The primary causes can be classified into lymphatic leakage due to increased interstitial pressure and increased leakage of protein-rich fluids due to erosive or non-erosive gastrointestinal disorders. The diagnosis of protein-losing enteropathy should be considered in patients with chronic diarrhea and peripheral oedema. The diagnosis of protein-losing enteropathy is most commonly based on the determination of fecal alpha-1 antitrypsin clearance. Most protein-losing enteropathy cases are the result of either lymphatic obstruction or a variety of gastrointestinal disorders and cardiac diseases, while primary intestinal lymphangiectasia (Waldmann's disease) is less common. Treatment of protein-losing enteropathy targets the underlying disease but also includes dietary modification, such as high-protein and low-fat diet along with medium-chain triglyceride supplementation. PMID:25618488

  15. Learning about Proteins

    MedlinePlus

    ... body, and protecting you from disease. All About Amino Acids When you eat foods that contain protein, the ... called amino (say: uh-MEE-no) acids. The amino acids then can be reused to make the proteins ...

  16. Hydrodynamic effects in proteins

    NASA Astrophysics Data System (ADS)

    Szymczak, Piotr; Cieplak, Marek

    2011-01-01

    Experimental and numerical results pertaining to flow-induced effects in proteins are reviewed. Special emphasis is placed on shear-induced unfolding and on the role of solvent mediated hydrodynamic interactions in the conformational transitions in proteins.

  17. Hydrodynamic effects in proteins.

    PubMed

    Szymczak, Piotr; Cieplak, Marek

    2011-01-26

    Experimental and numerical results pertaining to flow-induced effects in proteins are reviewed. Special emphasis is placed on shear-induced unfolding and on the role of solvent mediated hydrodynamic interactions in the conformational transitions in proteins. PMID:21406855

  18. Understanding protein folding: small proteins in silico.

    PubMed

    Zimmermann, Olav; Hansmann, Ulrich H E

    2008-01-01

    Recent improvements in methodology and increased computer power now allow atomistic computer simulations of protein folding. We briefly review several advanced Monte Carlo algorithms that have contributed to this development. Details of folding simulations of three designed mini proteins are shown. Adding global translations and rotations has allowed us to handle multiple chains and to simulate the aggregation of six beta-amyloid fragments. In a different line of research we have developed several algorithms to predict local features from sequence. In an outlook we sketch how such biasing could extend the application spectrum of Monte Carlo simulations to structure prediction of larger proteins. PMID:18036571

  19. Imaging Protein-protein Interactions in vivo

    PubMed Central

    Seegar, Tom; Barton, William

    2010-01-01

    Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using Förster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface. PMID:20972411

  20. Texturized dairy proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dairy proteins are amenable to structural modifications induced by high temperature, shear and moisture; in particular, whey proteins can change conformation to new unfolded states. The change in protein state is a basis for creating new foods. The dairy products, nonfat dried milk (NDM), whey prote...

  1. Palmitoylation of Hedgehog proteins.

    PubMed

    Buglino, John A; Resh, Marilyn D

    2012-01-01

    Hedgehog (Hh) proteins are secreted signaling proteins that contain amide-linked palmitate at the N-terminus and cholesterol at the C-terminus. Palmitoylation of Hh proteins is critical for effective long- and short-range signaling. The palmitoylation reaction occurs during transit of Hh through the secretory pathway, most likely in the lumen of the ER. Attachment of palmitate to Hh proteins is independent of cholesterol modification and autoprocessing and is catalyzed by Hhat (Hedgehog acyltransferase). Hhat is a member of the membrane bound O-acyltransferase (MBOAT) family, a subgroup of multipass membrane proteins that catalyze transfer of fatty acyl groups to lipids and proteins. Several classes of secreted proteins have recently been shown to be substrates for MBOAT acyltransferases, including Hh proteins and Spitz (palmitoylated by Hhat), Wg/Wnt proteins (modified with palmitate and/or palmitoleate by Porcupine) and ghrelin (octanoylated by ghrelin O-acyltransferase). These findings highlight protein fatty acylation as a mechanism that not only influences membrane binding of intracellular proteins but also regulates the signaling range and efficacy of secreted proteins. PMID:22391306

  2. Protein electrophoresis - serum

    MedlinePlus

    Normal value ranges are: Total protein: 6.4 to 8.3 g/dL (grams per deciliter) Albumin: 3.5 to 5.0 g/dL Alpha-1 ... Decreased total protein may indicate: Abnormal loss of protein from the digestive tract or the inability of the digestive tract ...

  3. CSF total protein

    MedlinePlus

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 mg/dL. Note: mg/dL = ...

  4. CSF myelin basic protein

    MedlinePlus

    CSF myelin basic protein is a test to measure the level of myelin basic protein (MBP) in the cerebrospinal fluid (CSF). The CSF ... less than 4 ng/mL of myelin basic protein in the CSF. Normal value ranges may vary ...

  5. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  6. Destabilized bioluminescent proteins

    DOEpatents

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  7. Modeling Protein Self Assembly

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck; Hull, Elizabeth

    2004-01-01

    Understanding the structure and function of proteins is an important part of the standards-based science curriculum. Proteins serve vital roles within the cell and malfunctions in protein self assembly are implicated in degenerative diseases. Experience indicates that this topic is a difficult one for many students. We have found that the concept…

  8. Protein - Which is Best?

    PubMed

    Hoffman, Jay R; Falvo, Michael J

    2004-09-01

    Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function) are also reviewed. Key PointsHigher protein needs are seen in athletic populations.Animal proteins is an important source of protein, however potential health concerns do exist from a diet of protein

  9. The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts*

    PubMed Central

    Wang, Zhen; Anderson, Nicholas Scott; Benning, Christoph

    2013-01-01

    Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus. PMID:23297418

  10. Telomere-binding protein regulates the chromosome ends through the interaction with histone deacetylases in Arabidopsis thaliana.

    PubMed

    Lee, Won Kyung; Cho, Myeon Haeng

    2016-06-01

    Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes. Many telomere-binding proteins bind to telomeric repeat sequences and further generate T-loops in animals. However, it is not clear if they regulate telomere organization using epigenetic mechanisms and how the epigenetic molecules are involved in regulating the telomeres. Here, we show direct interactions between the telomere-binding protein, AtTRB2 and histone deacetylases, HDT4 and HDA6, in vitro and in vivo AtTRB2 mediates the associations of HDT4 and HDA6 with telomeric repeats. Telomere elongation is found in AtTRB2, HDT4 and HDA6 mutants over generations, but also in met1 and cmt3 DNA methyltransferases mutants. We also characterized HDT4 as an Arabidopsis H3K27 histone deacetylase. HDT4 binds to acetylated peptides at residue K27 of histone H3 in vitro, and deacetylates this residue in vivo Our results suggest that AtTRB2 also has a role in the regulation of telomeric chromatin as a possible scaffold protein for recruiting the epigenetic regulators in Arabidopsis, in addition to its telomere binding and length regulation activity. Our data provide evidences that epigenetic molecules associate with telomeres by direct physical interaction with telomere-binding proteins and further regulate homeostasis of telomeres in Arabidopsis thaliana. PMID:26857545

  11. Telomere-binding protein regulates the chromosome ends through the interaction with histone deacetylases in Arabidopsis thaliana

    PubMed Central

    Lee, Won Kyung; Cho, Myeon Haeng

    2016-01-01

    Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes. Many telomere-binding proteins bind to telomeric repeat sequences and further generate T-loops in animals. However, it is not clear if they regulate telomere organization using epigenetic mechanisms and how the epigenetic molecules are involved in regulating the telomeres. Here, we show direct interactions between the telomere-binding protein, AtTRB2 and histone deacetylases, HDT4 and HDA6, in vitro and in vivo. AtTRB2 mediates the associations of HDT4 and HDA6 with telomeric repeats. Telomere elongation is found in AtTRB2, HDT4 and HDA6 mutants over generations, but also in met1 and cmt3 DNA methyltransferases mutants. We also characterized HDT4 as an Arabidopsis H3K27 histone deacetylase. HDT4 binds to acetylated peptides at residue K27 of histone H3 in vitro, and deacetylates this residue in vivo. Our results suggest that AtTRB2 also has a role in the regulation of telomeric chromatin as a possible scaffold protein for recruiting the epigenetic regulators in Arabidopsis, in addition to its telomere binding and length regulation activity. Our data provide evidences that epigenetic molecules associate with telomeres by direct physical interaction with telomere-binding proteins and further regulate homeostasis of telomeres in Arabidopsis thaliana. PMID:26857545

  12. Protein crystallization with paper

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  13. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  14. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  15. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-11-29

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  16. Selective Precipitation of Proteins.

    PubMed

    Matulis, Daumantas

    2016-01-01

    Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described. PMID:26836410

  17. Forces Stabilizing Proteins

    PubMed Central

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

    2014-01-01

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. 1. Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH2– group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. 2. The burial of nonpolar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. 3. Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. 4. The contribution of hydrogen bonds to protein stability is strongly context dependent. 5. Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 6. Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. 7. Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability. PMID:24846139

  18. Mechanism of protein decarbonylation.

    PubMed

    Wong, Chi-Ming; Marcocci, Lucia; Das, Dividutta; Wang, Xinhong; Luo, Haibei; Zungu-Edmondson, Makhosazane; Suzuki, Yuichiro J

    2013-12-01

    Ligand/receptor stimulation of cells promotes protein carbonylation that is followed by the decarbonylation process, which might involve thiol-dependent reduction (C.M. Wong et al., Circ. Res. 102:301-318; 2008). This study further investigated the properties of this protein decarbonylation mechanism. We found that the thiol-mediated reduction of protein carbonyls is dependent on heat-labile biologic components. Cysteine and glutathione were efficient substrates for decarbonylation. Thiols decreased the protein carbonyl content, as detected by 2,4-dinitrophenylhydrazine, but not the levels of malondialdehyde or 4-hydroxynonenal protein adducts. Mass spectrometry identified proteins that undergo thiol-dependent decarbonylation, which include peroxiredoxins. Peroxiredoxin-2 and -6 were carbonylated and subsequently decarbonylated in response to the ligand/receptor stimulation of cells. siRNA knockdown of glutaredoxin inhibited the decarbonylation of peroxiredoxin. These results strengthen the concept that thiol-dependent decarbonylation defines the kinetics of protein carbonylation signaling. PMID:24044890

  19. Protein solubility modeling

    NASA Technical Reports Server (NTRS)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.

    1999-01-01

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

  20. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  1. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  2. Phage display of proteins.

    PubMed

    Kościelska, K; Kiczak, L; Kasztura, M; Wesołowska, O; Otlewski, J

    1998-01-01

    In recent years the phage display approach has become an increasingly popular method in protein research. This method enables the presentation of large peptide and protein libraries on the surface of phage particles from which molecules of desired functional property(ies) can be rapidly selected. The great advantage of this method is a direct linkage between an observed phenotype and encapsulated genotype, which allows fast determination of selected sequences. The phage display approach is a powerful tool in generating highly potent biomolecules, including: search for specific antibodies, determining enzyme specificity, exploring protein-protein and protein-DNA interactions, minimizing proteins, introducing new functions into different protein scaffolds, and searching sequence space of protein folding. In this article many examples are given to illustrate that this technique can be used in different fields of protein science. The phage display has a potential of the natural evolution and its possibilities are far beyond rational prediction. Assuming that we can design the selection agents and conditions we should be able to engineer any desired protein function or feature. PMID:9918498

  3. GTP cyclohydrolase I feedback regulatory protein-dependent and -independent inhibitors of GTP cyclohydrolase I.

    PubMed

    Yoneyama, T; Wilson, L M; Hatakeyama, K

    2001-04-01

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates the feedback inhibition of GTP cyclohydrolase I activity by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) through protein complex formation. Since guanine and BH4 have a common pyrimidine ring structure, we examined the inhibitory effect of guanine and its analogs on the enzyme activity. Guanine, 8-hydroxyguanine, 8-methylguanine, and 8-bromoguanine inhibited the enzyme activity in a GFRP-dependent and pH-dependent manner and induced complex formation between GTP cyclohydrolase I and GFRP. The type of inhibition by this group is a mixed type. All these properties were shared with BH4. In striking contrast, inhibition by 8-azaguanine and 8-mercaptoguanine was GFRP-independent and pH-independent. The type of inhibition by 8-azaguanine and 8-mercaptoguanine was a competitive type. The two compounds did not induce complex formation between the enzyme and GFRP. These results demonstrate that guanine compounds of the first group bind to the BH4-binding site of the GTP cyclohydrolase I/GFRP complex, whereas 8-azaguanine and 8-mercaptoguanine bind to the active site of the enzyme. Finally, the possible implications in Lesch-Nyhan syndrome and Parkinson diseases of the inhibition of GTP cyclohydrolase I by guanine and 8-hydroxyguanine are discussed. PMID:11361142

  4. A redox-regulated chloroplast protein phosphatase binds to starch diurnally and functions in its accumulation.

    PubMed

    Sokolov, Lubomir N; Dominguez-Solis, Jose R; Allary, Anne-Laure; Buchanan, Bob B; Luan, Sheng

    2006-06-20

    Starch is the ultimate storage molecule formed in the photosynthetic fixation of carbon dioxide by chloroplasts. Starch accumulates during the day and is degraded at night to intermediates that are exported to heterotrophic organs. The mechanism by which diurnal cycles control the transitory biosynthesis and degradation of chloroplast starch has long remained a mystery. We now report evidence that a dual-specificity protein phosphatase, DSP4, binds to starch granules during the day and dissociates at night. Disruption of the DSP4 gene resulted in a dramatic increase in the level of starch in mutant Arabidopsis plants. Moreover, although composition was apparently unchanged, the morphology of the starch granule was significantly altered compared to the wild type counterpart. Two regulatory factors linked to light (i.e., pH and redox status) changed both the activity and the starch-binding capacity of DSP4. The results further revealed that DSP4 represents a major fraction of granule-bound phosphatase activity during the day but not at night. Our study suggests that DSP4 acts as a bridge between light-induced redox changes and protein phosphorylation in the regulation of starch accumulation. PMID:16772378

  5. Energy design for protein-protein interactions

    PubMed Central

    Ravikant, D. V. S.; Elber, Ron

    2011-01-01

    Proteins bind to other proteins efficiently and specifically to carry on many cell functions such as signaling, activation, transport, enzymatic reactions, and more. To determine the geometry and strength of binding of a protein pair, an energy function is required. An algorithm to design an optimal energy function, based on empirical data of protein complexes, is proposed and applied. Emphasis is made on negative design in which incorrect geometries are presented to the algorithm that learns to avoid them. For the docking problem the search for plausible geometries can be performed exhaustively. The possible geometries of the complex are generated on a grid with the help of a fast Fourier transform algorithm. A novel formulation of negative design makes it possible to investigate iteratively hundreds of millions of negative examples while monotonically improving the quality of the potential. Experimental structures for 640 protein complexes are used to generate positive and negative examples for learning parameters. The algorithm designed in this work finds the correct binding structure as the lowest energy minimum in 318 cases of the 640 examples. Further benchmarks on independent sets confirm the significant capacity of the scoring function to recognize correct modes of interactions. PMID:21842951

  6. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  7. Energy design for protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Ravikant, D. V. S.; Elber, Ron

    2011-08-01

    Proteins bind to other proteins efficiently and specifically to carry on many cell functions such as signaling, activation, transport, enzymatic reactions, and more. To determine the geometry and strength of binding of a protein pair, an energy function is required. An algorithm to design an optimal energy function, based on empirical data of protein complexes, is proposed and applied. Emphasis is made on negative design in which incorrect geometries are presented to the algorithm that learns to avoid them. For the docking problem the search for plausible geometries can be performed exhaustively. The possible geometries of the complex are generated on a grid with the help of a fast Fourier transform algorithm. A novel formulation of negative design makes it possible to investigate iteratively hundreds of millions of negative examples while monotonically improving the quality of the potential. Experimental structures for 640 protein complexes are used to generate positive and negative examples for learning parameters. The algorithm designed in this work finds the correct binding structure as the lowest energy minimum in 318 cases of the 640 examples. Further benchmarks on independent sets confirm the significant capacity of the scoring function to recognize correct modes of interactions.

  8. Protein-protein docking with backbone flexibility.

    PubMed

    Wang, Chu; Bradley, Philip; Baker, David

    2007-10-19

    Computational protein-protein docking methods currently can create models with atomic accuracy for protein complexes provided that the conformational changes upon association are restricted to the side chains. However, it remains very challenging to account for backbone conformational changes during docking, and most current methods inherently keep monomer backbones rigid for algorithmic simplicity and computational efficiency. Here we present a reformulation of the Rosetta docking method that incorporates explicit backbone flexibility in protein-protein docking. The new method is based on a "fold-tree" representation of the molecular system, which seamlessly integrates internal torsional degrees of freedom and rigid-body degrees of freedom. Problems with internal flexible regions ranging from one or more loops or hinge regions to all of one or both partners can be readily treated using appropriately constructed fold trees. The explicit treatment of backbone flexibility improves both sampling in the vicinity of the native docked conformation and the energetic discrimination between near-native and incorrect models. PMID:17825317

  9. Outer membrane protein purification.

    PubMed

    Arigita, C; Jiskoot, W; Graaf, M R; Kersten, G F

    2001-01-01

    The major outer membrane proteins (OMPs) from Neisseria meningitidis, which are expressed at high levels, are subdivided in five classes based on molecular weight (1,2) (see Table 1). Table 1 Major Meningococcal Outer-Membrane Proteins Outer-membrane proteins Name Molecular maass Function/characteristics Class 1 PorA 44-47 kDa Porin Class 2/3 PorB 37-42 kDa Porin Class 4 Rmp Reductionmodifiableprotein, unknown Class 5 Opa 26-30 kDa Adhesion,opacity protein Opc 25 kDa Invasion, opacity protein Iron-regulated proteins Mirp 37 kDa Iron acquisition (?);majoriron-regulatedprotein FrpB 70 kDa Ferric enterobactin receptor (also FetA) Adapted from ref. (1). PMID:21336748

  10. Mechanisms Regulating Protein Localization.

    PubMed

    Bauer, Nicholas C; Doetsch, Paul W; Corbett, Anita H

    2015-10-01

    Cellular functions are dictated by protein content and activity. There are numerous strategies to regulate proteins varying from modulating gene expression to post-translational modifications. One commonly used mode of regulation in eukaryotes is targeted localization. By specifically redirecting the localization of a pool of existing protein, cells can achieve rapid changes in local protein function. Eukaryotic cells have evolved elegant targeting pathways to direct proteins to the appropriate cellular location or locations. Here, we provide a general overview of these localization pathways, with a focus on nuclear and mitochondrial transport, and present a survey of the evolutionarily conserved regulatory strategies identified thus far. We end with a description of several specific examples of proteins that exploit localization as an important mode of regulation. PMID:26172624

  11. Electrophoretic separation of proteins.

    PubMed

    Chakavarti, Bulbul; Chakavarti, Deb

    2008-01-01

    Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). PMID:19066548

  12. Biofilm Matrix Proteins

    PubMed Central

    Fong, Jiunn N. C.; Yildiz, Fitnat H.

    2015-01-01

    Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this chapter, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation. PMID:26104709

  13. Antimicrobial proteins: From old proteins, new tricks.

    PubMed

    Smith, Valerie J; Dyrynda, Elisabeth A

    2015-12-01

    This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. The review further considers proteins or protein fragments from crustaceans that have antimicrobial properties but are more usually associated with other biological functions, or are derived from such proteins. It discusses how these unconventional AMPs might be generated at, or delivered to, sites of infection and how they might contribute to crustacean host defence in vivo. It also highlights recent work that is starting to reveal the extent of multi-functionality displayed by some decapod AMPs, particularly their participation in other aspects of host protection. Examples of such activities include proteinase inhibition, phagocytosis, antiviral activity and haematopoiesis. PMID:26320628

  14. Elastic proteins and elastomeric protein alloys.

    PubMed

    Aghaei-Ghareh-Bolagh, Behnaz; Mithieux, Suzanne M; Weiss, Anthony S

    2016-06-01

    The elastomeric proteins elastin and resilin have been used extensively in the fabrication of biomaterials for tissue engineering applications due to their unique mechanical and biological properties. Tropoelastin is the soluble monomer component of elastin. Tropoelastin and resilin are both highly elastic with high resilience, substantial extensibility, high durability and low energy loss, which makes them excellent candidates for the fabrication of elastic tissues that demand regular and repetitive movement like the skin, lung, blood vessels, muscles and vocal folds. Combinations of these proteins with silk fibroin further enhance their biomechanical and biological properties leading to a new class of protein alloy materials with versatile properties. In this review, the properties of tropoelastin-based and resilin-based biomaterials with and without silk are described in concert with examples of their applications in tissue engineering. PMID:26780495

  15. Principles of Flexible Protein-Protein Docking

    PubMed Central

    Andrusier, Nelly; Mashiach, Efrat; Nussinov, Ruth; Wolfson, Haim J.

    2008-01-01

    Treating flexibility in molecular docking is a major challenge in cell biology research. Here we describe the background and the principles of existing flexible protein-protein docking methods, focusing on the algorithms and their rational. We describe how protein flexibility is treated in different stages of the docking process: in the preprocessing stage, rigid and flexible parts are identified and their possible conformations are modeled. This preprocessing provides information for the subsequent docking and refinement stages. In the docking stage, an ensemble of pre-generated conformations or the identified rigid domains may be docked separately. In the refinement stage, small-scale movements of the backbone and side-chains are modeled and the binding orientation is improved by rigid-body adjustments. For clarity of presentation, we divide the different methods into categories. This should allow the reader to focus on the most suitable method for a particular docking problem. PMID:18655061

  16. DNA prime-protein boost using subtype consensus Env was effective in eliciting neutralizing antibody responses against subtype BC HIV-1 viruses circulating in China

    PubMed Central

    Zhang, Mingshun; Zhang, Lu; Zhang, Chunhua; Hong, Kunxue; Shao, Yiming; Huang, Zuhu; Wang, Shixia; Lu, Shan

    2012-01-01

    Previously, we have shown that DNA prime-protein boost is effective in eliciting neutralizing antibodies (NAb) against randomly selected HIV-1 isolates. Given the genetic diversity of HIV-1 viruses and the unique predominant subtypes in different geographic regions, it is critical to test the DNA prime-protein boost approach against circulating viral isolates in key HIV endemic areas. In the current study, the same DNA prime-protein boost vaccine was used as in previous studies to investigate the induction of NAb responses against HIV-1 clade BC, a major subtype circulating in China. A codon optimized gp120-BC DNA vaccine, based on the consensus envelope (Env) antigen sequence of clade BC, was constructed and a stable CHO cell line expressing the same consensus BC gp120 protein was produced. The immunogenicity of this consensus gp120-BC was examined in New Zealand White rabbits by either DNA prime-protein boost or protein alone vaccination approaches. High levels of Env-specific antibody responses were elicited by both approaches. However, DNA prime-protein boost but not the protein alone immune sera contained significant levels of NAb against pseudotyped viruses expressing HIV-1 BC Env antigens. Furthermore, high frequencies of CD4 binding site-targeted antibodies were found in the DNA prime- protein boost rabbit sera indicating that the positive NAb may be the result of antibodies against conformationally sensitive epitopes on HIV-1 Env. The findings support that DNA prime-protein boost was effective in eliciting NAb against a key HIV-1 virus subtype in China. This result may lead to the development of regional HIV vaccines through this approach. PMID:23111170

  17. Computer Models of Proteins

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Dr. Marc Pusey (seated) and Dr. Craig Kundrot use computers to analyze x-ray maps and generate three-dimensional models of protein structures. With this information, scientists at Marshall Space Flight Center can learn how proteins are made and how they work. The computer screen depicts a proten structure as a ball-and-stick model. Other models depict the actual volume occupied by the atoms, or the ribbon-like structures that are crucial to a protein's function.

  18. Pressure cryocooling protein crystals

    DOEpatents

    Kim, Chae Un; Gruner, Sol M.

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  19. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  20. Protein oxidation and peroxidation.

    PubMed

    Davies, Michael J

    2016-04-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established. PMID:27026395

  1. Protein oxidation and peroxidation

    PubMed Central

    Davies, Michael J.

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established. PMID:27026395

  2. Protein-protein interactions as drug targets.

    PubMed

    Skwarczynska, Malgorzata; Ottmann, Christian

    2015-10-01

    Modulation of protein-protein interactions (PPIs) is becoming increasingly important in drug discovery and chemical biology. While a few years ago this 'target class' was deemed to be largely undruggable an impressing number of publications and success stories now show that targeting PPIs with small, drug-like molecules indeed is a feasible approach. Here, we summarize the current state of small-molecule inhibition and stabilization of PPIs and review the active molecules from a structural and medicinal chemistry angle, especially focusing on the key examples of iNOS, LFA-1 and 14-3-3. PMID:26510391

  3. Prediction of protein-protein interactions based on protein-protein correlation using least squares regression.

    PubMed

    Huang, De-Shuang; Zhang, Lei; Han, Kyungsook; Deng, Suping; Yang, Kai; Zhang, Hongbo

    2014-01-01

    In order to transform protein sequences into the feature vectors, several works have been done, such as computing auto covariance (AC), conjoint triad (CT), local descriptor (LD), moran autocorrelation (MA), normalized moreaubroto autocorrelation (NMB) and so on. In this paper, we shall adopt these transformation methods to encode the proteins, respectively, where AC, CT, LD, MA and NMB are all represented by '+' in a unified manner. A new method, i.e. the combination of least squares regression with '+' (abbreviated as LSR(+)), will be introduced for encoding a protein-protein correlation-based feature representation and an interacting protein pair. Thus there are totally five different combinations for LSR(+), i.e. LSRAC, LSRCT, LSRLD, LSRMA and LSRNMB. As a result, we combined a support vector machine (SVM) approach with LSR(+) to predict protein-protein interactions (PPI) and PPI networks. The proposed method has been applied on four datasets, i.e. Saaccharomyces cerevisiae, Escherichia coli, Homo sapiens and Caenorhabditis elegans. The experimental results demonstrate that all LSR(+) methods outperform many existing representative algorithms. Therefore, LSR(+) is a powerful tool to characterize the protein-protein correlations and to infer PPI, whilst keeping high performance on prediction of PPI networks. PMID:25059329

  4. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  5. PIC: Protein Interactions Calculator.

    PubMed

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside. PMID:17584791

  6. Glycolipid transfer proteins

    PubMed Central

    Brown, Rhoderick E.; Mattjus, Peter

    2007-01-01

    Glycolipid transfer proteins (GLTPs) are small (24 kD), soluble, ubiquitous proteins characterized by their ability to accelerate the intermembrane transfer of glycolipids in vitro. GLTP specificity encompasses both sphingoid- and glycerol-based glycolipids, but with a strict requirement that the initial sugar residue be beta-linked to the hydrophobic lipid backbone. The 3D protein structures of GLTP reveal liganded structures with unique lipid binding modes. The biochemical properties of GLTP action at the membrane surface have been studied rather comprehensively, but the biological role of GLTP remains enigmatic. What is clear is that GLTP differs distinctly from other known glycolipid-binding proteins, such as nonspecific lipid transfer proteins, lysosomal sphingolipid activator proteins, lectins, lung surfactant proteins as well as other lipid binding/transfer proteins. Based on the unique conformational architecture that targets GLTP to membranes and enables glycolipid binding, GLTP is now considered the prototypical and founding member of a new protein superfamily in eukaryotes. PMID:17320476

  7. Engineering therapeutic protein disaggregases.

    PubMed

    Shorter, James

    2016-05-15

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Alzheimer's disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. PMID:27255695

  8. Biomolecular membrane protein crystallization

    NASA Astrophysics Data System (ADS)

    Reddy Bolla, Jani; Su, Chih-Chia; Yu, Edward W.

    2012-07-01

    Integral membrane proteins comprise approximately 30% of the sequenced genomes, and there is an immediate need for their high-resolution structural information. Currently, the most reliable approach to obtain these structures is X-ray crystallography. However, obtaining crystals of membrane proteins that diffract to high resolution appears to be quite challenging, and remains a major obstacle in structural determination. This brief review summarizes a variety of methodologies for use in crystallizing these membrane proteins. Hopefully, by introducing the available methods, techniques, and providing a general understanding of membrane proteins, a rational decision can be made about now to crystallize these complex materials.

  9. Self assembling proteins

    DOEpatents

    Yeates, Todd O.; Padilla, Jennifer; Colovos, Chris

    2004-06-29

    Novel fusion proteins capable of self-assembling into regular structures, as well as nucleic acids encoding the same, are provided. The subject fusion proteins comprise at least two oligomerization domains rigidly linked together, e.g. through an alpha helical linking group. Also provided are regular structures comprising a plurality of self-assembled fusion proteins of the subject invention, and methods for producing the same. The subject fusion proteins find use in the preparation of a variety of nanostructures, where such structures include: cages, shells, double-layer rings, two-dimensional layers, three-dimensional crystals, filaments, and tubes.

  10. Consensus protein design.

    PubMed

    Porebski, Benjamin T; Buckle, Ashley M

    2016-07-01

    A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino acids. Here, we review the consensus design approach, its theoretical underpinnings, successes, limitations and challenges, as well as providing a detailed guide to its application in protein engineering. PMID:27274091

  11. Engineering therapeutic protein disaggregases

    PubMed Central

    Shorter, James

    2016-01-01

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. PMID:27255695

  12. Cellulose synthase interacting protein

    PubMed Central

    Somerville, Chris

    2010-01-01

    Cellulose is the most abundant biopolymer on earth. The great abundance of cellulose places it at the forefront as a primary source of biomass for renewable biofuels. However, the knowledge of how plant cells make cellulose remains very rudimentary. Cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes. The only known components of cellulose synthase complexes are cellulose synthase (CESA) proteins until the recent identification of a novel component. CSI1, which encodes CESA interacting protein 1 (CSI1) in Arabidopsis. CSI1, as the first non-CESA proteins associated with cellulose synthase complexes, opens up many opportunities. PMID:21150290

  13. Consensus protein design

    PubMed Central

    Porebski, Benjamin T.; Buckle, Ashley M.

    2016-01-01

    A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino acids. Here, we review the consensus design approach, its theoretical underpinnings, successes, limitations and challenges, as well as providing a detailed guide to its application in protein engineering. PMID:27274091

  14. Acanthamoeba castellanii STAT protein.

    PubMed

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups. PMID:25338074

  15. Expression and biological activity of genetic fusions between MalE, the maltose binding protein from Escherichia coli and portions of CD4, the T-cell receptor of the AIDS virus.

    PubMed

    Clément, J M; Jehanno, M; Popescu, O; Saurin, W; Hofnung, M

    1996-11-01

    Hybrid molecules between MalE, the periplasmic maltose binding protein of Escherichia coli, and CD4, the human T-lymphocyte receptor for the AIDS virus HIV, have been constructed and purified. We show that CD4 can be fused as multiple repeats to both ends of a single MalE molecule. Hybrid proteins are exported into the periplasm of bacteria, bind monoclonal antibodies directed against CD4, bind HIV gp160, and inhibit HIV binding to CD4+ cells. MalE has been used as a scaffold to graft portions of CD4. Deletion analysis allowed to define a minimal structural domain which folds in a way which is compatible with its biological activity. This minimal part was used to design compact hybrid molecules in which CD4 was inserted internally into MalE. PMID:8936594

  16. Ultrafiltration of pegylated proteins

    NASA Astrophysics Data System (ADS)

    Molek, Jessica R.

    There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG--protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with nonoptimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein via primary amines on the lysine residues. A simple model was developed for the reaction kinetics, which was used to explore the effect of reaction conditions and mode of operation on the distribution of PEGylated products. The effective size of the PEGylated proteins was evaluated using size exclusion chromatography, with appropriate correlations developed for the size in terms of the molecular weight of the native protein and attached PEG. The electrophoretic mobility of the PEGylated proteins were evaluated by capillary electrophoresis with the data in good agreement with a simple model accounting for the increase in protein size and the reduction in the number of protonated amine

  17. Protein metabolism and requirements.

    PubMed

    Biolo, Gianni

    2013-01-01

    Skeletal muscle adaptation to critical illness includes insulin resistance, accelerated proteolysis, and increased release of glutamine and the other amino acids. Such amino acid efflux from skeletal muscle provides precursors for protein synthesis and energy fuel to the liver and to the rapidly dividing cells of the intestinal mucosa and the immune system. From these adaptation mechanisms, severe muscle wasting, glutamine depletion, and hyperglycemia, with increased patient morbidity and mortality, may ensue. Protein/amino acid nutrition, through either enteral or parenteral routes, plays a pivotal role in treatment of metabolic abnormalities in critical illness. In contrast to energy requirement, which can be accurately assessed by indirect calorimetry, methods to determine individual protein/amino acid needs are not currently available. In critical illness, a decreased ability of protein/amino acid intake to promote body protein synthesis is defined as anabolic resistance. This abnormality leads to increased protein/amino acid requirement and relative inefficiency of nutritional interventions. In addition to stress mediators, immobility and physical inactivity are key determinants of anabolic resistance. The development of mobility protocols in the intensive care unit should be encouraged to enhance the efficacy of nutrition. In critical illness, protein/amino acid requirement has been defined as the intake level associated with the lowest rate of catabolism. The optimal protein-sparing effects in patients receiving adequate energy are achieved when protein/amino acids are administered at rates between 1.3 and 1.5 g/kg/day. Extra glutamine supplementation is required in conditions of severe systemic inflammatory response. Protein requirement increases during hypocaloric feeding and in patients with acute renal failure on continuous renal replacement therapy. Evidence suggests that receiving adequate protein/amino acid intake may be more important than achieving

  18. Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling.

    PubMed

    Santos, Celio X C; Hafstad, Anne D; Beretta, Matteo; Zhang, Min; Molenaar, Chris; Kopec, Jola; Fotinou, Dina; Murray, Thomas V; Cobb, Andrew M; Martin, Daniel; Zeh Silva, Maira; Anilkumar, Narayana; Schröder, Katrin; Shanahan, Catherine M; Brewer, Alison C; Brandes, Ralf P; Blanc, Eric; Parsons, Maddy; Belousov, Vsevelod; Cammack, Richard; Hider, Robert C; Steiner, Roberto A; Shah, Ajay M

    2016-02-01

    Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine-threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species-generating NADPH oxidase-4 (Nox4) is induced downstream of ATF4, binds to a PP1-targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4-regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia-reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4-GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress. PMID:26742780

  19. Preserved bioactivity and tunable release of a SDF1-GPVI bi-specific protein using photo-crosslinked PEGda hydrogels.

    PubMed

    Schesny, Marianne K; Monaghan, Michael; Bindermann, Andrea H; Freund, Désirée; Seifert, Martina; Eble, Johannes A; Vogel, Sebastian; Gawaz, Meinrad P; Hinderer, Svenja; Schenke-Layland, Katja

    2014-08-01

    Chemokine-induced stem cell recruitment is a promising strategy for post myocardial infarction treatment. Injection of stromal cell-derived factor 1 (SDF1) has been shown to attract bone marrow-derived progenitor cells (BMPCs) from the blood that have the potential to differentiate into cardiovascular cells, which support angiogenesis, enabling the improvement of myocardial function. SDF1-GPVI bi-specific protein contains a glycoprotein VI (GPVI)-domain that serves as an anchor for collagen type I (Col I) and III, which are exposed in the wall of injured vasculature. In this study, we generated a cytocompatible hydrogel via photo-crosslinking of poly(ethylene glycol) diacrylate that serves as a reservoir for SDF1-GPVI. Controlled and sustained release of SDF1-GPVI was demonstrated over a period of 7 days. Release features were modifiable depending on the degree of the crosslinking density. Functionality of the GPVI-domain was investigated using a GPVI-binding ELISA to Col I. Activity of the SDF1-domain was tested for its CXCR4 binding potential. Preserved functionality of SDF1-GPVI bi-specific protein after photo-crosslinking and controllable release was successfully demonstrated in vitro supporting the implementation of this drug delivery system as a powerful tool for therapeutic protein delivery in the treatment of cardiovascular ischemic disease. PMID:24875761

  20. Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells.

    PubMed

    Yu, Nuo; Signorile, Luca; Basu, Sreya; Ottema, Sophie; Lebbink, Joyce H G; Leslie, Kris; Smal, Ihor; Dekkers, Dick; Demmers, Jeroen; Galjart, Niels

    2016-07-11

    The microtubule (MT) cytoskeleton forms a dynamic filamentous network that is essential for many processes, including mitosis, cell polarity and shape, neurite outgrowth and migration, and ciliogenesis [1, 2]. MTs are built up of α/β-tubulin heterodimers, and their dynamic behavior is in part regulated by tubulin-associated proteins (TAPs). Here we describe a novel system to study mammalian tubulins and TAPs. We co-expressed equimolar amounts of triple-tagged α-tubulin and β-tubulin using a 2A "self-cleaving" peptide and isolated functional fluorescent tubulin dimers from transfected HEK293T cells with a rapid two-step approach. We also produced two mutant tubulins that cause brain malformations in tubulinopathy patients [3]. We then applied a paired mass-spectrometry-based method to identify tubulin-binding proteins in HEK293T cells and describe both novel and known TAPs. We find that CKAP5 and the CLASPs, which are MT plus-end-tracking proteins with TOG(L)-domains [4], bind tubulin efficiently, as does the Golgi-associated protein GCC185, which interacts with the CLASPs [5]. The N-terminal TOGL domain of CLASP1 contributes to tubulin binding and allows CLASP1 to function as an autonomous MT-growth-promoting factor. Interestingly, mutant tubulins bind less well to a number of TAPs, including CLASPs and GCC185, and incorporate less efficiently into cellular MTs. Moreover, expression of these mutants in cells impairs several MT-growth-related processes involving TAPs. Thus, stable tubulin-TAP interactions regulate MT nucleation and growth in cells. Combined, our results provide a resource for investigating tubulin interactions and functions and widen the spectrum of tubulin-related disease mechanisms. PMID:27291054

  1. Binding Efficiency of Protein-Protein Complexes

    PubMed Central

    Day, Eric S.; Cote, Shaun M.; Whitty, Adrian

    2012-01-01

    We examine the relationship between binding affinity and interface size for reversible protein-protein interactions (PPI), using cytokines from the tumor necrosis factor (TNF) superfamily and their receptors as a test case. Using surface plasmon resonance, we measured single-site binding affinities for the large receptor TNFR1 binding to its ligands TNFα (KD = 1.4 ± 0.4 nM) and lymphotoxin-α (KD = 50 ± 10 nM), and also for the small receptor Fn14 binding to TWEAK (KD = 70 ± 10 nM). We additionally assembled data for all other TNF/TNFR family complexes for which reliable single site binding affinities have been reported. We used these values to calculate the binding efficiency – defined as binding energy per Å2 of surface area buried at the contact interface – for the nine of these complexes for which co-crystal structures are available, and compared the results to those for a set of 144 protein-protein complexes with published affinity values. The results show that the most efficient PPI complexes generate ~20 cal.mol−1/Å2 of binding energy. A minimum contact area of ~500 Å2 is required for a stable complex, required to generate sufficient interaction energy to pay the entropic cost of co-localizing two proteins from 1 M solution. The most compact and efficient TNF/TNFR complex was BAFF/BR3, which achieved ~80% of the maximum achievable binding efficiency. Other small receptors also gave high binding efficiencies, while the larger receptors generated only 44-49% of this limit despite interacting primarily through just a single small domain. The results provide new insight into how much binding energy can be generated by a PPI interface of a given size, and establish a quantitative method to predict how large a natural or engineered contact interface must be to achieve a given level of binding affinity. PMID:23088250

  2. Synthesis of Lipidated Proteins.

    PubMed

    Mejuch, Tom; Waldmann, Herbert

    2016-08-17

    Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented. PMID:27444727

  3. Protein Attachment on Nanodiamonds.

    PubMed

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery. PMID:25815400

  4. Poxviral Ankyrin Proteins

    PubMed Central

    Herbert, Michael H.; Squire, Christopher J.; Mercer, Andrew A

    2015-01-01

    Multiple repeats of the ankyrin motif (ANK) are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range. PMID:25690795

  5. Proteins and Amino Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the most abundant substances in living organisms and cells. All proteins are constructed from the same twenty amino acids that are linked together by covalent bonds. Shorter chains of two or more amino acids can be linked by covalent bonds to form polypeptides. There are twenty amino...

  6. Proteins and glasses

    SciTech Connect

    Frauenfelder, H.

    1997-12-31

    The structure, the energy landscape, and the dynamics of proteins and glasses are similar. Both types of systems display characteristic nonexponential time dependencies of relaxation phenomena. Experiments suggest that both, proteins and glasses, are heterogeneous and that this fact causes the observed time dependence. This result is discussed in terms of the rough energy landscape characteristic of complex systems.

  7. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  8. The AVIT protein family

    PubMed Central

    Kaser, Alexandra; Winklmayr, Martina; Lepperdinger, Günther; Kreil, Günther

    2003-01-01

    Homologues of a protein originally isolated from snake venom and frog skin secretions are present in many vertebrate species. They contain 80–90 amino acids, 10 of which are cysteines with identical spacing. Various names have been given to these proteins, such as mamba intestinal protein 1 (MIT1), Bv8 (Bombina variegata molecular mass ∼8 kDa), prokineticins and endocrine-gland vascular endothelial growth factor (EG-VEGF). Their amino-terminal sequences are identical, and so we propose that the sequence of their first four residues, AVIT, is used as a name for this family. From a comparison of the sequences, two types of AVIT proteins can be discerned. These proteins seem to be distributed widely in mammalian tissues and are known to bind to G-protein-coupled receptors. Members of this family have been shown to stimulate contraction of the guinea pig ileum, to cause hyperalgesia after injection into rats and to be active as specific growth factors. Moreover, the messenger RNA level of one of these AVIT proteins changes rhythmically in the region of the brain known as the suprachiasmatic nucleus. This shows that members of this new family of small proteins are involved in diverse biological processes. PMID:12728244

  9. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  10. Manipulating and Visualizing Proteins

    SciTech Connect

    Simon, Horst D.

    2003-12-05

    ProteinShop Gives Researchers a Hands-On Tool for Manipulating, Visualizing Protein Structures. The Human Genome Project and other biological research efforts are creating an avalanche of new data about the chemical makeup and genetic codes of living organisms. But in order to make sense of this raw data, researchers need software tools which let them explore and model data in a more intuitive fashion. With this in mind, researchers at Lawrence Berkeley National Laboratory and the University of California, Davis, have developed ProteinShop, a visualization and modeling program which allows researchers to manipulate protein structures with pinpoint control, guided in large part by their own biological and experimental instincts. Biologists have spent the last half century trying to unravel the ''protein folding problem,'' which refers to the way chains of amino acids physically fold themselves into three-dimensional proteins. This final shape, which resembles a crumpled ribbon or piece of origami, is what determines how the protein functions and translates genetic information. Understanding and modeling this geometrically complex formation is no easy matter. ProteinShop takes a given sequence of amino acids and uses visualization guides to help generate predictions about the secondary structures, identifying alpha helices and flat beta strands, and the coil regions that bind them. Once secondary structures are in place, researchers can twist and turn these pre-configurations until they come up with a number of possible tertiary structure conformations. In turn, these are fed into a computationally intensive optimization procedure that tries to find the final, three-dimensional protein structure. Most importantly, ProteinShop allows users to add human knowledge and intuition to the protein structure prediction process, thus bypassing bad configurations that would otherwise be fruitless for optimization. This saves compute cycles and accelerates the entire process, so