Penicillin-Binding Protein 1A, 2B, and 2X Alterations in Canadian Isolates of Penicillin-Resistant Streptococcus pneumoniae

PubMed Central

Nichol, Kimberly A.; Zhanel, George G.; Hoban, Daryl J.

2002-01-01

Alterations within the penicillin-binding domain of penicillin-binding protein (PBP) genes pbp1a, pbp2b, and pbp2x were determined for 15 Canadian isolates of Streptococcus pneumoniae. All penicillin-nonsusceptible S. pneumoniae isolates showed a variety of PBP 2X substitutions and contained a Thr445-Ala change after the PBP 2B SSN motif. Only isolates for which penicillin MICs were ≥0.5 μg/ml had PBP 1A alterations near the STMK and SRN motifs. Sequence analysis revealed identical PBP 1A, PBP 2B, and PBP 2X substitution patterns among all isolates for which penicillin MICs were ≥1 μg/ml. PMID:12234855

Importance of adhesins in the recurrence of pharyngeal infections caused by Streptococcus pyogenes.

PubMed

Wozniak, Aniela; Scioscia, Natalia; Geoffroy, Enrique; Ponce, Iván; García, Patricia

2017-04-01

Pharyngo-amygdalitis is the most common infection caused by Streptococcus pyogenes (S. pyogenes). Reinfection with strains of different M types commonly occurs. However, a second infection with a strain of the same M type can still occur and is referred to as recurrence. We aimed to assess whether recurrence of S. pyogenes could be associated to erythromycin resistance, biofilm formation or surface adhesins like fibronectin-binding proteins and pilus proteins, both located in the fibronectin-binding, collagen-binding, T-antigen (FCT) region. We analyed clinical isolates of S. pyogenes obtained from children with multiple positive cultures of throat swabs. We analysed potential associations between M types, clonal patterns, biofilm production and FCT types with their capacity of producing a recurrent infection. We genetically defined recurrence as an infection with the same M type (same strain) and reinfection as an infection with a different M type. No differences were observed between recurrent and reinfection isolates in relation to erythromycin resistance, presence and number of domains of prtF1 gene, and biofilm formation capacity; the only significant difference was the higher frequency of FCT-4 type among recurrent isolates. However, when all the factors that could contribute to recurrence (erythromycin resistance, biofilm production, presence of prtF1 gene and FCT-4 type) were analysed together, we observed that recurrent isolates have a higher number of factors than reinfection isolates. Recurrence seems not to be associated with biofilm formation. However, pili and fibronectin-binding proteins could be associated with recurrence because FCT-4 isolates which harbour two fibronectin-binding proteins are more frequent among recurrent isolates.

Factor H binds to the hypervariable region of many Streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence.

PubMed

Gustafsson, Mattias C U; Lannergård, Jonas; Nilsson, O Rickard; Kristensen, Bodil M; Olsen, John E; Harris, Claire L; Ufret-Vincenty, Rafael L; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

2013-01-01

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.

Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

PubMed Central

Kristensen, Bodil M.; Olsen, John E.; Harris, Claire L.; Ufret-Vincenty, Rafael L.; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

2013-01-01

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems. PMID:23637608

Identification of cytosolic peroxisome proliferator binding protein as a member of the heat shock protein HSP70 family.

PubMed Central

Alvares, K; Carrillo, A; Yuan, P M; Kawano, H; Morimoto, R I; Reddy, J K

1990-01-01

Clofibrate and many of its structural analogues induce proliferation of peroxisomes in the hepatic parenchymal cells of rodents and certain nonrodent species including primates. This induction is tissue specific, occurring mainly in the liver parenchymal cells and to a lesser extent in the kidney cortical epithelium. The induction of peroxisomes is associated with a predictable pleiotropic response, characterized by hepatomegaly, and increased activities and mRNA levels of certain peroxisomal enzymes. Using affinity chromatography, we had previously isolated a protein that binds to clofibric acid. We now show that this protein is homologous with the heat shock protein HSP70 family by analysis of amino acid sequences of isolated peptides from trypsin-treated clofibric acid binding protein and by cross-reactivity with a monoclonal antibody raised against the conserved region of the 70-kDa heat shock proteins. The clofibric acid-Sepharose column could bind HSP70 proteins isolated from various species, which could then be eluted with either clofibric acid or ATP. Conversely, when a rat liver cytosol containing multiple members of the HSP70 family was passed through an ATP-agarose column, and eluted with clofibric acid, only P72 (HSC70) was eluted. These results suggest that clofibric acid, a peroxisome proliferator, preferentially interacts with P72 at or near the ATP binding site. Images PMID:2371272

A small cellulose binding domain protein (CBD1) is highly variable in the nonbinding amino terminus

USDA-ARS?s Scientific Manuscript database

The small cellulose binding domain protein CBD1 is tightly bound to the cellulosic cell wall of the plant pathogenic stramenophile Phytophthora infestans. Transgene expression of the protein in plants has also demonstrated binding to plant cell walls. A study was undertaken using 47 isolates of P. ...

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida.

PubMed

Hoover, G J; el-Mowafi, A; Simko, E; Kocal, T E; Ferguson, H W; Hayes, M A

1998-07-01

In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A. salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated. Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE. Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms. A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37). This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A. salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Characterization studies on cadmium-mycophosphatin from the mushroom Agaricus macrosporus.

PubMed Central

Meisch, H U; Schmitt, J A

1986-01-01

A low molecular weight Cd-binding phosphoglycoprotein, cadmium-mycophosphatin, has been isolated from the mushroom Agaricus macrosporus. This protein has a molecular weight of 12,000 dalton and contains no sulfur but a high amount of acid amino acids (Glu, Asp), and carbohydrates (glucose, galactose). Cadmium-mycophosphatin has an isoelectric point less than pH 2, binds cadmium with a dissociation constant of KD = 1.59 X 10 M (pKD = 6.8) and is saturated with 13.5 mole Cd/mole, all Cd-binding sites being equivalent. It is suggested that Cd is bound by phosphoserine groups, similar relations being known from calcium-binding proteins in animals. From A. macrosporus four other low-molecular weight glycoproteins have been isolated which contain sulfur and bind cadmium and copper. The biological significance of these Cd-binding proteins is discussed. PMID:3709455

Distinctive receptor binding properties of the surface glycoprotein of a natural feline leukemia virus isolate with unusual disease spectrum.

PubMed

Bolin, Lisa L; Chandhasin, Chandtip; Lobelle-Rich, Patricia A; Albritton, Lorraine M; Levy, Laura S

2011-05-13

Feline leukemia virus (FeLV)-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU) as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor. Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB) to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect. The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E) when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread in vivo, or by facilitating entry into a novel target cell with a low receptor density.

Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

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

Turley, E.A.; Moore, D.; Hayden, L.J.

1987-06-02

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weightmore » range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.« less

Purification of Proteins Fused to Maltose-Binding Protein.

PubMed

Lebendiker, Mario; Danieli, Tsafi

2017-01-01

Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.

Characterization studies on cadmium-mycophosphatin from the mushroom Agaricus macrosporus

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

Meisch, H.U.; Schmitt, J.A.

A low molecular weight Cd-binding phosphoglycoprotein, cadmium-mycophosphatin, has been isolated from the mushroom Agaricus macrosporus. This protein has a molecular weight of 12,000 dalton and contains no sulfur but a high amount of acid amino acids (Glu, Asp), and carbohydrates (glucose, galactose). Cadmium-mycophosphatin has an isoelectric point less than pH 2, binds cadmium with a dissociation constant of K/sub D/ = 1.59 x 10 M (pK/sub D/ = 6.8) and is saturated with 13.5 mole Cd/mole, all Cd-binding sites being equivalent. It is suggested that Cd is bound by phosphoserine groups, similar relations being known from calcium-binding proteins in animals.more » From A. macrosporus four other low-molecular weight glycoproteins have been isolated which contain sulfur and bind cadmium and copper. The biological significance of these Cd-binding proteins is discussed.« less

A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

NASA Technical Reports Server (NTRS)

Reddy, A. S.; Reddy, V. S.; Golovkin, M.

2000-01-01

Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

Metal-binding proteins as metal pollution indicators

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

Hennig, H.F.

1986-03-01

The fact that metal-binding proteins are a consequence of elevated metal concentration in organisms is well known. What has been overlooked is that the presence of these proteins provides a unique opportunity to reformulate the criteria of metal pollution. The detoxification effect of metal-binding proteins in animals from polluted areas has been cited, but there have been only very few studies relating metal-binding proteins to pollution. This lack is due partly to the design of most experiments, which were aimed at isolation of metal-binding proteins and hence were of too short duration to allow for correlation to adverse physiological effectsmore » on the organism. In this study metal-binding proteins were isolated and characterized from five different marine animals (rock lobster, Jasus lalandii; hermit crab, Diogenes brevirostris; sandshrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis; and limpet, Patella granularis). These animals were kept under identical metal-enriched conditions, hence eliminating differences in method and seasons. The study animals belonged to different phyla; varied in size, mass, age, behavior, food requirements and life stages; and accumulated metals at different rates. It is possible to link unseasonal moulting in crustacea, a known physiological effect due to a metal-enriched environment, to the production of the metal-binding protein without evidence of obvious metal body burden. Thus a new concept of pollution is defined: the presence of metal-binding proteins confirms toxic metal pollution. This concept was then tested under field conditions in the whelk Bullia digitalis and in metal-enriched grass.« less

Isolation and characterization of fatty acid binding protein in the liver of the nurse shark, Ginglymostoma cirratum.

PubMed

Bass, N M; Manning, J A; Luer, C A

1991-01-01

1. A 14.5 kDa fatty acid binding protein was isolated from the liver of the nurse shark, Ginglymostoma cirratum. 2. Purified shark liver FABP (pI = 5.4) bound oleic acid at a single site with an affinity similar to that of mammalian FABP. 3. The apparent size, pI and amino acid composition of shark liver FABP indicate a close structural relationship between this protein and mammalian heart FABP.

Heterogeneous RNA-binding protein M4 is a receptor for carcinoembryonic antigen in Kupffer cells.

PubMed

Bajenova, O V; Zimmer, R; Stolper, E; Salisbury-Rowswell, J; Nanji, A; Thomas, P

2001-08-17

Here we report the isolation of the recombinant cDNA clone from rat macrophages, Kupffer cells (KC) that encodes a protein interacting with carcinoembryonic antigen (CEA). To isolate and identify the CEA receptor gene we used two approaches: screening of a KC cDNA library with a specific antibody and the yeast two-hybrid system for protein interaction using as a bait the N-terminal part of the CEA encoding the binding site. Both techniques resulted in the identification of the rat heterogeneous RNA-binding protein (hnRNP) M4 gene. The rat ortholog cDNA sequence has not been previously described. The open reading frame for this gene contains a 2351-base pair sequence with the polyadenylation signal AATAAA and a termination poly(A) tail. The mRNA shows ubiquitous tissue expression as a 2.4-kilobase transcript. The deduced amino acid sequence comprised a 78-kDa membrane protein with 3 putative RNA-binding domains, arginine/methionine/glutamine-rich C terminus and 3 potential membrane spanning regions. When hnRNP M4 protein is expressed in pGEX4T-3 vector system in Escherichia coli it binds (125)I-labeled CEA in a Ca(2+)-dependent fashion. Transfection of rat hnRNP M4 cDNA into a non-CEA binding mouse macrophage cell line p388D1 resulted in CEA binding. These data provide evidence for a new function of hnRNP M4 protein as a CEA-binding protein in Kupffer cells.

A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

PubMed

Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

2015-02-01

Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. © 2014 Society for Laboratory Automation and Screening.

Human CRISP-3 binds serum alpha(1)B-glycoprotein across species.

PubMed

Udby, Lene; Johnsen, Anders H; Borregaard, Niels

2010-04-01

CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments. We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing. We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG. It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera. Copyright 2009 Elsevier B.V. All rights reserved.

Isolation and partial purification of cadmium-binding protein from roots of the grass Agrostis gigantea

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

Rauser, W.E.

1984-04-01

A cadmium-binding protein was isolated from roots of the grass Agrostis gigantea Roth. Heat-stable proteins were chromatographed on the anion exchanger QAE-Sephadex A-25. The major cadmium fraction was purified further by gel filtration of Sephadex G-75 in 1 molar KCI buffer. The resulting protein preparation was light brown, had an apparent molecular weight of 3700, contained 29% cysteine and close to 4 gram atoms cadmium/mole. The cadmium:cysteine ratio was l:2.7. Spectroscopic measurments indicated cadmium-thiolate coordination. The roots produced the metallothionein-like protein when they were exposed to cadmium for 7 days.

Uptake of oleate by isolated rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut

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

Schwieterman, W.; Sorrentino, D.; Potter, B.J.

1988-01-01

A portion of the hepatocellular uptake of nonesterified long-chain fatty acids is mediated by a specific 40-kDa plasma membrane fatty acid binding protein, which has also been isolated from the gut. To investigate whether a similar transport process exists in other tissues with high transmembrane fatty acid fluxes, initial rates (V/sub O/) of (/sup 3/H)-oleate uptake into isolated rat adipocytes were studied as a function of the concentration of unbound (/sup 3/H)oleate in the medium. V/sub O/ reached a maximum as the concentration of unbound oleate was increased and was significantly inhibited both by phloretin and by prior incubation ofmore » the cells with Pronase. A rabbit antibody to the rat liver plasma membrane fatty acid binding protein inhibited adipocyte fatty acid uptake by up to 63% in dose-dependent fashion. Inhibition was noncompetitive; at an immunoglobulin concentration of 250 ..mu..g/ml V/sub max/ was reduced from 2480 /plus minus/ 160 to 1870 /plus minus/ 80 pmol/min per 5 /times/ 10/sup 4/ adipocytes, with no change in K/sub m/. A basic kDa adipocyte plasma membrane fatty acid binding protein, isolated from crude adipocyte plasma membrane fractions, reacted strongly in both agar gel diffusion and electrophoretic blots with the antibody raised against the corresponding hepatic plasma membrane protein. These data indicate that the uptake of oleate by rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut.« less

The Determination of Vitamin D-Dependent Calcium Binding Protein in Chick Intesting: An Undergraduate Biochemistry Laboratory Experiment.

ERIC Educational Resources Information Center

Lessard, George M.

1980-01-01

Described is an experiment used in an undergraduate biochemistry laboratory involving inducing rickets in chicks and correlating the disease to a reduction in vitamin D-dependent calcium binding protein. Techniques involved are hormone induction, protein isolation, and radioisotope methodology. (Author/DS)

Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family.

PubMed

Nallapareddy, Sreedhar R; Weinstock, George M; Murray, Barbara E

2003-03-01

A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.

Hyperdiversity of Genes Encoding Integral Light-Harvesting Proteins in the Dinoflagellate Symbiodinium sp

PubMed Central

Boldt, Lynda; Yellowlees, David; Leggat, William

2012-01-01

The superfamily of light-harvesting complex (LHC) proteins is comprised of proteins with diverse functions in light-harvesting and photoprotection. LHC proteins bind chlorophyll (Chl) and carotenoids and include a family of LHCs that bind Chl a and c. Dinophytes (dinoflagellates) are predominantly Chl c binding algal taxa, bind peridinin or fucoxanthin as the primary carotenoid, and can possess a number of LHC subfamilies. Here we report 11 LHC sequences for the chlorophyll a-chlorophyll c 2-peridinin protein complex (acpPC) subfamily isolated from Symbiodinium sp. C3, an ecologically important peridinin binding dinoflagellate taxa. Phylogenetic analysis of these proteins suggests the acpPC subfamily forms at least three clades within the Chl a/c binding LHC family; Clade 1 clusters with rhodophyte, cryptophyte and peridinin binding dinoflagellate sequences, Clade 2 with peridinin binding dinoflagellate sequences only and Clades 3 with heterokontophytes, fucoxanthin and peridinin binding dinoflagellate sequences. PMID:23112815

Purification and partial characterization of a cadmium-binding protein from the liver of rainbow trout (Onchorynchus mykiss).

PubMed Central

Mullins, J E; Fredrickson, R A; Fuentealba, I C; Markham, R J

1999-01-01

This study describes the isolation and partial characterization of a low molecular weight (approximately 14 kDa), cadmium-binding protein from rainbow trout (Onchorynchus mykiss) liver. Rainbow trout were injected intraperitoneally with 3.5 mg/kg cadmium chloride (total body dose) twice weekly for 3 wk. Livers were removed and a cadmium-binding protein was isolated. Monoclonal antibodies produced against this protein were used in the affinity purification process. Amino acid analysis showed the protein contained 3.8 mol% cysteine, 3.5 mol% phenylalanine, 2.2 mol% tyrosine and 1.9 mol% histidine. The low cysteine content suggests that it was distinct from metallothionein. The monoclonal antibodies were also used to identify the protein in liver homogenates from both cadmium-exposed and control fish and in the testes of cadmium-exposed mice lacking the gene for both metallothionein-1 and metallothionein-II. The compound identified in this study represents a non-metallothionein cadmium-binding protein that appears to be highly conserved. Images Figure 1. Figure 2. Figure 3. Figure 4. PMID:10534000

Proteoform-specific protein binding of small molecules in complex matrices

USDA-ARS?s Scientific Manuscript database

Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original ...

Allelic variation of polymorphic locus lytB, encoding a choline-binding protein, from streptococci of the mitis group.

PubMed

Moscoso, Miriam; Obregón, Virginia; López, Rubens; García, José L; García, Ernesto

2005-12-01

The choline-binding protein LytB, an N-acetylglucosaminidase of Streptococcus pneumoniae, is the key enzyme for daughter cell separation and is believed to play a critical pathogenic role, facilitating bacterial spreading during infection. Because of these peculiarities LytB is a putative vaccine target. To determine the extent of LytB polymorphism, the lytB alleles from seven typical, clinical pneumococcal isolates of various serotypes and from 13 additional streptococci of the mitis group (12 atypical pneumococci and the Streptococcus mitis type strain) were sequenced. Sequence alignment showed that the main differences among alleles were differences in the number of repeats (range, 12 to 18) characteristic of choline-binding proteins. These differences were located in the region corresponding to repeats 11 to 17. Typical pneumococcal strains contained either 14, 16, or 18 repeats, whereas all of the atypical isolates except strains 1283 and 782 (which had 14 and 16 repeats, respectively) and the S. mitis type strain had only 12 repeats; atypical isolate 10546 turned out to be a DeltalytB mutant. We also found that there are two major types of alternating repeats in lytB, which encode 21 and 23 amino acids. Choline-binding proteins are linked to the choline-containing cell wall substrate through choline residues at the interface of two consecutive choline-binding repeats that create a choline-binding site. The observation that all strains contained an even number of repeats suggests that the duplication events that gave rise to the choline-binding repeats of LytB involved two repeats simultaneously, an observation that is in keeping with previous crystallographic data. Typical pneumococcal isolates usually grew as diplococci, indicating that an active LytB enzyme was present. In contrast, most atypical isolates formed long chains of cells that did not disperse after addition of purified LytB, suggesting that in these strains chains were produced through mechanisms unrelated to LytB.

Monoclonal antibodies of African swine fever virus: antigenic differences among field virus isolates and viruses passaged in cell culture.

PubMed Central

García-Barreno, B; Sanz, A; Nogal, M L; Viñuela, E; Enjuanes, L

1986-01-01

An analysis of the binding properties of a collection of monoclonal antibodies to African swine fever virus particles showed that virus field isolates passaged in porcine macrophages changed antigenically more than a strain of a cell-adapted virus passaged in Vero cells. From seven clones isolated from the spleen of a field-infected pig, we found four clones that had the same antigenic properties, one clone that had large changes in proteins p150 and p27 and small changes in proteins p37 and p14, and two clones that had minor changes in proteins p150 and p27, respectively. An analysis of the binding properties of the monoclonal antibodies to 23 field isolates from Africa, Europe, and America showed that the African isolates differed among themselves more than the European and the American isolates; in this study we found changes in 8 of the 10 virus proteins tested. The most variable proteins in the African isolates were p150, p27, p14, and p12. In contrast to the African isolates, protein p12 from the non-African viruses did not change. The clustering of the field virus isolates in six antigenic homology groups indicated the existence of a complex variety of African swine fever virus serotypes. PMID:2422393

Genome-Wide Cell Type-Specific Mapping of In Vivo Chromatin Protein Binding Using an FLP-Inducible DamID System in Drosophila.

PubMed

Pindyurin, Alexey V

2017-01-01

A thorough study of the genome-wide binding patterns of chromatin proteins is essential for understanding the regulatory mechanisms of genomic processes in eukaryotic nuclei, including DNA replication, transcription, and repair. The DNA adenine methyltransferase identification (DamID) method is a powerful tool to identify genomic binding sites of chromatin proteins. This method does not require fixation of cells and the use of specific antibodies, and has been used to generate genome-wide binding maps of more than a hundred different proteins in Drosophila tissue culture cells. Recent versions of inducible DamID allow performing cell type-specific profiling of chromatin proteins even in small samples of Drosophila tissues that contain heterogeneous cell types. Importantly, with these methods sorting of cells of interest or their nuclei is not necessary as genomic DNA isolated from the whole tissue can be used as an input. Here, I describe in detail an FLP-inducible DamID method, namely generation of suitable transgenic flies, activation of the Dam transgenes by the FLP recombinase, isolation of DNA from small amounts of dissected tissues, and subsequent identification of the DNA binding sites of the chromatin proteins.

Fimbrial subunit protein FaeG expressed in transgenic tobacco inhibits the binding of F4ac enterotoxigenic Escherichia coli to porcine enterocytes.

PubMed

Joensuu, Jussi J; Kotiaho, Mirkka; Riipi, Tero; Snoeck, Veerle; Palva, E Tapio; Teeri, Teemu H; Lång, Hannu; Cox, Eric; Goddeeris, Bruno M; Niklander-Teeri, Viola

2004-06-01

Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed. Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants. In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein. Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment. For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast. The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein. The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions. Plant-produced FaeG proved to be stable up to 2 h under these conditions. The binding and inhibition properties were tested with isolated piglet villi. These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner. Thus, the first two prerequisites for the development of an oral vaccine have been met.

[Glutamate-binding membrane proteins from human platelets].

PubMed

Gurevich, V S; Popov, Iu G; Gorodinskiĭ, A I; Dambinova, S A

1991-09-01

Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.

Cloning of a heavy-metal-binding protein derived from activated-sludge microorganisms.

PubMed

Sano, Daisuke; Myojo, Ken; Omura, Tatsuo

2006-09-01

A gene of the heavy-metal-binding protein (HMBP) was newly isolated from a genetic DNA library of activated-sludge microorganisms. HMBP was produced by transformed Escherichia coli, and the copper-binding ability of HMBP was confirmed. HMBP derived from activated sludge could be available as heavy metal adsorbents in water and wastewater treatments.

Identification of Lectins from Metastatic Cancer Cells through Magnetic Glyconanoparticles

PubMed Central

Kavunja, Herbert W.; Voss, Patricia G.

2016-01-01

Cancer cells can have characteristic carbohydrate binding properties. Previously, it was shown that a highly metastatic melanoma cell line B16F10 bound to galacto-side-functionalized nanoparticles much stronger than the corresponding less metastatic B16F1 cells. To better understand the carbohydrate binding properties of cancer cells, herein, we report the isolation and characterization of endogenous galactose binding proteins from B16F10 cells using magnetic glyconanoparticles. The galactose-coated magnetic glyconanoparticles could bind with lectins present in the cells and be isolated through magnet-mediated separation. Through Western blot and mass spectrometry, the arginine/serine rich splicing factor Sfrs1 was identified as a galactose-selective endogenous lectin, overexpressed in B16F10 cells, compared with B16F1 cells. In addition, galactin-3 was found in higher amounts in B16F10 cells. Finally, the glyconanoparticles exhibited a superior efficiency in lectin isolation, from both protein mixtures and live cells, than the corresponding more traditional microparticles functionalized with carbohydrates. Thus, the magnetic glyconanoparticles present a useful tool for discovery of endogenous lectins, as well as binding partners of lectins, without prior knowledge of protein identities. PMID:27110035

Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

PubMed Central

SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

2016-01-01

SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

Effect of enzymatic deamidation of soy protein by protein-glutaminase on the flavor-binding properties of the protein under aqueous conditions.

PubMed

Suppavorasatit, Inthawoot; Cadwallader, Keith R

2012-08-15

The effect of the enzymatic deamidation by protein-glutaminase (PG) on flavor-binding properties of soy protein isolate (SPI) under aqueous conditions was evaluated by a modified equilibrium dialysis (ultrafiltration) technique. Binding parameters, such as number of binding sites (n) and binding constants (K), were derived from Klotz plots. The partial deamidation of SPI by PG (43.7% degree of deamidation) decreased overall flavor-binding affinity (nK) at 25 °C for both vanillin and maltol by approximately 9- and 4-fold, respectively. The thermodynamic parameters of binding indicated that the flavor-protein interactions were spontaneous (negative ΔG°) and that the driving force of the interactions shifted from entropy to enthalpy driven as a result of deamidation. Deamidation of soy protein caused a change in the mechanism of binding from hydrophobic interactions or covalent bonding (Schiff base formation) to weaker van der Waals forces or hydrogen bonding.

A protein isolated from human oviductal tissue in vitro secretion, identified as human lactoferrin, interacts with spermatozoa and oocytes and modulates gamete interaction.

PubMed

Zumoffen, C M; Gil, R; Caille, A M; Morente, C; Munuce, M J; Ghersevich, S A

2013-05-01

Is lactoferrin (LF) (detected in oviductal secretion) able to bind to oocytes and sperm and modulate gamete interaction? LF binds to zona pellucida (ZP) and spermatozoa (depending upon the capacitation stage and acrosome status) and inhibits gamete interaction in vitro. Proteins from human oviductal tissue secretion modulate gamete interaction and parameters of sperm function in vitro and some of them bind to sperm, but they remain to be isolated and identified. Proteins were isolated from human oviductal tissue secretion using their sperm membrane binding ability. One of the isolated proteins was identified as human LF and immunolocalized in tubal tissues. LF expression was analyzed in native oviductal fluid and oviduct epithelial cells (at different phases of the menstrual cycle: proliferative, periovulatory and secretory). In addition, the LF binding sites on spermatozoa (at different capacitation and acrosome reaction stages) and on ZP and the dose-dependent effect of LF on gamete interaction were investigated. All experiments were performed at least three times. Tubal tissues obtained from premenopausal patients (scheduled for hysterectomy, n = 23) were cultured in DMEM/Ham's F12 medium and conditioned media (CM) were collected. Motile spermatozoa were obtained by swim-up from normozoospermic semen samples from healthy donors (n = 4). An affinity chromatography with sperm membrane extracts was used to isolate proteins from CM. Isolated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophresis and further identified by nano liquid chromatography tandem mass spectrometry peptide sequencing. The presence of LF in oviductal tissue was investigated by immunohistochemistry and immunofluorescence and was detected in native oviductal fluid and oviduct epithelial cells homogenates by western blot. LF binding sites on gametes were investigated by incubating gametes with the protein coupled to fluorescein isothiocyanate (FITC). The acrosome reaction was assessed with Pisum sativum agglutinin conjugated with rhodamine. The effect of increasing concentrations of LF (0.1-100 µg/ml) on gamete interaction was evaluated by a sperm-ZP binding assay, using human oocytes donated by women undergoing IVF procedures. A protein isolated by the affinity column was identified as human LF. LF was immunolocalized in human oviductal tissue and detected in oviductal fluid and oviduct epithelial cell homogenates. In the latter case, LF expression was highest at the periovulatory phase of the menstrual cycle (P < 0.01). Different LF binding patterns were observed on spermatozoa depending upon capacitation stage and if the acrosome reaction had occurred. Unstained sperm were most prevalent before capacitation, but after incubation for 6 h under capacitating conditions and in acrosome-reacted sperm LF binding was observed, mainly localized in the equatorial segment and post-acrosomal region of the sperm head. LF binding studies on ZP showed homogenous staining. LF caused a dose-dependent significant inhibition of sperm-ZP interaction, and the effect was already significant (P < 0.01) with the lowest LF concentration used. This study has investigated the effect of LF only on human gamete interaction in vitro and thus has some limitations. Further investigations of the potential mechanisms involved in LF action both on gamete function in vitro and in vivo in animal models are needed to confirm the role of this protein in the reproductive process. The present data indicate that human oviductal LF expression is cycle dependent and inhibited gamete interaction in vitro. No previous data were available about potential direct effects of LF on gamete interaction. It could be thought that the protein is involved in the regulation of the reproductive process, perhaps contributing to prevent polyspermy. Thus, further research is needed to clarify the potential role of LF in the regulation of the fertilization process. This study was supported by grants from FONCYT (PICT 01095, S.A.G., M.J.M) and SECyT UNR (PIDBIO238, S.A.G). The authors have no conflict of interest to declare.

[Non-enzymatic glycosylation of dietary protein in vitro].

PubMed

Bednykh, B S; Evdokimov, I A; Sokolov, A I

2015-01-01

Non-enzymatic glycosylation of proteins, based on discovered by Mayarn reaction of carbohydrate aldehyde group with a free amino group of a protein molecule, is well known to experts in biochemistry of food industry. Generated brown solid in some cases give the product marketable qualities--crackling bread--in others conversely, worsen the product. The biological effects of far-advanced products of non-enzymatic protein glycosylation reaction have not been studied enough, although it was reported previously that they are not split by digestive enzymes and couldn't be absorbed by animals. The objective of this work was to compare the depth of glycosylation of different food proteins of animal and vegetable origin. The objects of the study were proteins of animal (casein, lactoglobulin, albumin) and vegetable (soy isolate, proteins of rice flour, buckwheat, oatmeal) origin, glucose and fructose were selected as glycosylation agents, exposure 15 days at 37 degrees C. Lactoglobulin was glycosylated to a lesser extent among the proteins of animal origin while protein of oatmeal was glycosylated in the least degree among vegetable proteins. Conversely, such proteins as casein and soya isolate protein bound rather large amounts of carbohydrates. Fructose binding with protein was generally higher than the binding of glucose. The only exception was a protein of oatmeal. When of glucose and fructose simultaneously presented in the incubation medium, glucose binding usually increased while binding of fructose, in contrast, reduced. According to the total amount of carbohydrate (mcg), which is able to attach a protein (mg) the studied food proteins located in the following order: albumin (38) > soy protein isolate (23) > casein (15,) > whey protein rice flour protein (6) > protein from buckwheat flour (3) > globulin (2) > protein of oatmeal (0.3). The results obtained are to be used to select the optimal combination of proteins and carbohydrates, in which the glycosylation during storage will be minimal.

Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

NASA Technical Reports Server (NTRS)

Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

1993-01-01

Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA

PubMed Central

Alspach, Elise; Stewart, Sheila A.

2016-01-01

Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014). PMID:27453911

Odorant-binding proteins from a primitive termite.

PubMed

Ishida, Yuko; Chiang, Vicky P; Haverty, Michael I; Leal, Walter S

2002-09-01

Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths.

Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2

PubMed Central

Kitahara, Kei; Kajiura, Akimasa; Sato, Neuza Satomi; Suzuki, Tsutomu

2007-01-01

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function. PMID:17553838

Isolation and characterization of a novel calmodulin-binding protein from potato

NASA Technical Reports Server (NTRS)

Reddy, Anireddy S N.; Day, Irene S.; Narasimhulu, S. B.; Safadi, Farida; Reddy, Vaka S.; Golovkin, Maxim; Harnly, Melissa J.

2002-01-01

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

Isolation and N-terminal sequencing of a novel cadmium-binding protein from Boletus edulis

NASA Astrophysics Data System (ADS)

Collin-Hansen, C.; Andersen, R. A.; Steinnes, E.

2003-05-01

A Cd-binding protein was isolated from the popular edible mushroom Boletus edulis, which is a hyperaccumulator of both Cd and Hg. Wild-growing samples of B. edulis were collected from soils rich in Cd. Cd radiotracer was added to the crude protein preparation obtained from ethanol precipitation of heat-treated cytosol. Proteins were then further separated in two consecutive steps; gel filtration and anion exchange chromatography. In both steps the Cd radiotracer profile showed only one distinct peak, which corresponded well with the profiles of endogenous Cd obtained by atomic absorption spectrophotometry (AAS). Concentrations of the essential elements Cu and Zn were low in the protein fractions high in Cd. N-terminal sequencing performed on the Cd-binding protein fractions revealed a protein with a novel amino acid sequence, which contained aromatic amino acids as well as proline. Both the N-terminal sequencing and spectrofluorimetric analysis with EDTA and ABD-F (4-aminosulfonyl-7-fluoro-2, 1, 3-benzoxadiazole) failed to detect cysteine in the Cd-binding fractions. These findings conclude that the novel protein does not belong to the metallothionein family. The results suggest a role for the protein in Cd transport and storage, and they are of importance in view of toxicology and food chemistry, but also for environmental protection.

A novel lipid transfer protein from the pea Pisum sativum: isolation, recombinant expression, solution structure, antifungal activity, lipid binding, and allergenic properties.

PubMed

Bogdanov, Ivan V; Shenkarev, Zakhar O; Finkina, Ekaterina I; Melnikova, Daria N; Rumynskiy, Eugene I; Arseniev, Alexander S; Ovchinnikova, Tatiana V

2016-04-30

Plant lipid transfer proteins (LTPs) assemble a family of small (7-9 kDa) ubiquitous cationic proteins with an ability to bind and transport lipids as well as participate in various physiological processes including defense against phytopathogens. They also form one of the most clinically relevant classes of plant allergens. Nothing is known to date about correlation between lipid-binding and IgE-binding properties of LTPs. The garden pea Pisum sativum is widely consumed crop and important allergenic specie of the legume family. This work is aimed at isolation of a novel LTP from pea seeds and characterization of its structural, functional, and allergenic properties. Three novel lipid transfer proteins, designated as Ps-LTP1-3, were found in the garden pea Pisum sativum, their cDNA sequences were determined, and mRNA expression levels of all the three proteins were measured at different pea organs. Ps-LTP1 was isolated for the first time from the pea seeds, and its complete amino acid sequence was determined. The protein exhibits antifungal activity and is a membrane-active compound that causes a leakage from artificial liposomes. The protein binds various lipids including bioactive jasmonic acid. Spatial structure of the recombinant uniformly (13)C,(15)N-labelled Ps-LTP1 was solved by heteronuclear NMR spectroscopy. In solution the unliganded protein represents the mixture of two conformers (relative populations ~ 85:15) which are interconnected by exchange process with characteristic time ~ 100 ms. Hydrophobic residues of major conformer form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ~1000 Å(3)). The minor conformer probably corresponds to the protein with the partially collapsed internal cavity. For the first time conformational heterogeneity in solution was shown for an unliganded plant lipid transfer protein. Heat denaturation profile and simulated gastrointestinal digestion assay showed that Ps-LTP1 displayed a high thermal and digestive proteolytic resistance proper for food allergens. The reported structural and immunological findings seem to describe Ps-LTP1 as potential cross-reactive allergen in LTP-sensitized patients, mostly Pru p 3(+) ones. Similarly to allergenic LTPs the potential IgE-binding epitope of Ps-LTP1 is located near the proposed entrance into internal cavity and could be involved in lipid-binding.

Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts.

PubMed

Kane, M A; Portanova, L B; Kelley, K; Holley, M; Ross, S E; Boose, D; Escobedo-Morse, A; Alvarado, B

1994-01-01

Swiss 3T3 cells contained substantial amounts of soluble and specific [125I]GRP binders. Like the membrane-associated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fractionation yielded different profiles, the M(rs) were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [125I]GRP. These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells.

Exploring DNA-binding Proteins with In Vivo Chemical Cross-linking and Mass Spectrometry

PubMed Central

Qiu, Haibo; Wang, Yinsheng

2009-01-01

DNA-binding proteins are very important constituents of proteomes of all species and play crucial roles in transcription, DNA replication, recombination, repair and other activities associated with DNA. Although a number of DNA-binding proteins have been identified, many proteins involved in gene regulation and DNA repair are likely still unknown because of their dynamic and/or weak interactions with DNA. In this report, we described an approach for the comprehensive identification of DNA-binding proteins with in vivo formaldehyde cross-linking and LC-MS/MS. DNA-binding proteins could be purified via the isolation of DNA-protein complexes and released from the complexes by reversing the cross-linking. By using this method, we were able to identify more than one hundred DNA-binding proteins, such as proteins involved in transcription, gene regulation, DNA replication and repair, and a large number of proteins which are potentially associated with DNA and DNA-binding proteins. This method should be generally applicable to the investigation of other nucleic acid-binding proteins, and hold great potential in the comprehensive study of gene regulation, DNA damage response and repair, as well as many other critical biological processes at proteomic level. PMID:19714816

Identification of pathogenic factors potentially involved in Staphylococcus aureus keratitis using proteomics.

PubMed

Khan, Shamila; Cole, Nerida; Hume, Emma B H; Garthwaite, Linda L; Nguyen-Khuong, Terry; Walsh, Bradley J; Willcox, Mark D P

2016-10-01

Staphylococcus is a leading cause of microbial keratitis, characterized by destruction of the cornea by bacterial exoproteins and host-associated factors. The aim of this study was to compare extracellular and cell-associated proteins produced by two different isolates of S. aureus, a virulent clinical isolate (Staph 38) and a laboratory strain (Staphylococcus aureus 8325-4) of weaker virulence in the mouse keratitis model. Proteins were analyzed using 2D polyacrylamide gel electrophoresis and identified by subsequent mass spectrometry. Activity of staphylococcal adhesins was assessed by allowing strains to bind to various proteins adsorbed onto polymethylmethacrylate squares. Thirteen proteins in the extracellular fraction and eight proteins in the cell-associated fractions after bacterial growth were produced in increased amounts in the clinical isolate Staph 38. Four of these proteins were S. aureus virulence factor adhesins, fibronectin binding protein A, staphopain, glyceraldehyde-3-phosphate dehydrogenase 2 and extracellular adherence protein. The clinical isolate Staph 38 adhered to a greater extent to all mammalian proteins tested, indicating the potential of the adhesins to be active on its surface. Other proteins with increased expression in Staph 38 included potential moonlighting proteins and proteins involved in transcription or translation. This is the first demonstration of the proteome of S. aureus isolates from keratitis. These results indicate that the virulent clinical isolate produces more potentially important virulence factors compared to the less virulent laboratory strain and these may be associated with the ability of a S. aureus strain to cause more severe keratitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Isolation and characterization of major histocompatibility complex class IIB genes from the nurse shark.

PubMed

Bartl, S; Weissman, I L

1994-01-04

The major histocompatibility complex (MHC) contains a set of linked genes which encode cell surface proteins involved in the binding of small peptide antigens for their subsequent recognition by T lymphocytes. MHC proteins share structural features and the presence and location of polymorphic residues which play a role in the binding of antigens. In order to compare the structure of these molecules and gain insights into their evolution, we have isolated two MHC class IIB genes from the nurse shark, Ginglymostoma cirratum. Two clones, most probably alleles, encode proteins which differ by 13 amino acids located in the putative antigen-binding cleft. The protein structure and the location of polymorphic residues are similar to their mammalian counterparts. Although these genes appear to encode a typical MHC protein, no T-cell-mediated responses have been demonstrated in cartilaginous fish. The nurse shark represents the most phylogenetically primitive organism in which both class IIA [Kasahara, M., Vazquez, M., Sato, K., McKinney, E.C. & Flajnik, M.F. (1992) Proc. Natl. Acad. Sci USA 89, 6688-6692] and class IIB genes, presumably encoding the alpha/beta heterodimer, have been isolated.

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

PubMed Central

Grange, T; de Sa, C M; Oddos, J; Pictet, R

1987-01-01

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis. Images PMID:2885805

Size-dependent protein segregation at membrane interfaces

PubMed Central

Schmid, Eva M; Bakalar, Matthew H; Choudhuri, Kaushik; Weichsel, Julian; Ann, HyoungSook; Geissler, Phillip L; Dustin, Michael L; Fletcher, Daniel A

2016-01-01

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane protein organization, such as E-cadherin enrichment in epithelial junctional complexes and CD45 exclusion from the signaling foci of immunological synapses. To isolate the role of protein size in these processes, we reconstituted membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between binding and non-binding proteins can dramatically alter their organization at membrane interfaces in the absence of active contributions from the cytoskeleton, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally-driven membrane height fluctuations that transiently limit access to the interface. This simple, sensitive, and highly effective means of passively segregating proteins has implications for signaling at cell-cell junctions and protein sorting at intracellular contact points between membrane-bound organelles. PMID:27980602

Binding of [51Cr]ethylenediaminetetraacetate to proteins of human plasma.

PubMed Central

Babiker, M M

1986-01-01

Binding of [51Cr]EDTA to human plasma proteins was investigated using chemical and chromatographic techniques of separation of the proteins and protein fractions. Total plasma proteins isolated with ethanol retained 12.95 +/- 0.46% of the initial plasma activity. Proteins separated by other precipitants retained about 16% of the initial radioactivity. Globulins exhibited the highest binding capacity for [51Cr]EDTA and retained about 11.7% of the initial plasma activity following chromatographic separation. This value represents about 70% of the radioactivity bound by the total proteins of the plasma. gamma-Globulins contributed most of the binding attributed to the globulins and retained about 8.7% of the initial [51Cr]EDTA activity. The repeatedly reported underestimation of the renal glomerular filtration rate when estimated as the clearance of [51Cr]EDTA could be adequately accounted for by the extent of binding of this marker to the plasma proteins. PMID:2427701

Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation.

PubMed

Bell, Thomas J; Eberwine, James

2015-01-01

RNA-binding proteins (RBPs) are essential regulatory proteins that control all modes of RNA processing and regulation. New experimental approaches to isolate these indispensable proteins under in vivo conditions are needed to advance the field of RBP biology. Historically, in vitro biochemical approaches to isolate RBP complexes have been useful and productive, but biological relevance of the identified RBP complexes can be imprecise or erroneous. Here we review an inventive experimental to isolate RBPs under the in vivo conditions. The method is called peptide nucleic acid (PNA)-assisted identification of RBP (PAIR) technology and it uses cell-penetrating peptides (CPPs) to deliver photo-activatible RBP-capture molecule to the cytoplasm of the live cells. The PAIR methodology provides two significant advantages over the most commonly used approaches: (1) it overcomes the in vitro limitation of standard biochemical approaches and (2) the PAIR RBP-capture molecule is highly selective and adaptable which allows investigators to isolate exon-specific RBP complexes. Most importantly, the in vivo capture conditions and selectivity of the RBP-capture molecule yield biologically accurate and relevant RBP data.

Analysis of the interaction between membrane proteins and soluble binding partners by surface plasmon resonance.

PubMed

Wu, Zht Cheng; de Keyzer, Jeanine; Kusters, Ilja; Driessen, Arnold J M

2013-01-01

The interaction between membrane proteins and their (protein) ligands is conventionally investigated by nonequilibrium methods such as co-sedimentation or pull-down assays. Surface Plasmon Resonance can be used to monitor such binding events in real-time using isolated membranes immobilized to a surface providing insights in the kinetics of binding under equilibrium conditions. This application provides a fast, automated way to detect interacting species and to determine the kinetics and affinity (Kd) of the interaction.

Identification of Collagen-Binding Proteins in Lactobacillus spp. with Surface-Enhanced Laser Desorption/Ionization–Time of Flight ProteinChip Technology

PubMed Central

Howard, Jeffrey C.; Heinemann, Christine; Thatcher, Bradley J.; Martin, Brian; Gan, Bing Siang; Reid, Gregor

2000-01-01

Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)–time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions. PMID:11010889

Quantitative cytochemistry of nuclear and cytoplasmic proteins using the Naphthol Yellow S and dinitrofluorobenzene staining methods.

PubMed

Tas, J; James, J

1981-09-01

The 'total protein staining' of biological specimens with the electrostatically binding Naphthol Yellow S or the covalently binding dinitrofluorobenzene must be interpreted as methods which yield data on the specific amino acid pool of the proteins concerned. Both dyes bind to certain free amino-acid side-chains, giving different dye--protein ratios for various proteins. In the presence of DNA, dinitrofluorobenzene stains all proteins present in cell nuclei, whereas Naphthol Yellow S only stains the majority of the non-histone proteins. When protein staining methods are combined with the Feulgen--Pararosanile (SO2) procedure for DNA, decreased Feulgen--DNA contents were measured in dinitrofluorobenzene-stained isolated nuclei and lymphocytes.

Polymorphisms in Fibronectin Binding Protein A of Staphylococcus Aureus are Associated with Infection of Cardiovascular Devices

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

Lower, Steven; Lamlertthon, Supaporn; Casillas-Ituarte, Nadia

Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a bio-film, a structured community of bacterial cells adherent to the surface of a solid substrate. Every bio-film begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated frommore » humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct bindingforce signature and had speci!c single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.« less

Immunoreactive Proteins of Bifidobacterium longum ssp. longum CCM 7952 and Bifidobacterium longum ssp. longum CCDM 372 Identified by Gnotobiotic Mono-Colonized Mice Sera, Immune Rabbit Sera and Non-immune Human Sera

PubMed Central

Górska, Sabina; Dylus, Ewa; Rudawska, Angelika; Brzozowska, Ewa; Srutkova, Dagmar; Schwarzer, Martin; Razim, Agnieszka; Kozakova, Hana; Gamian, Andrzej

2016-01-01

The Bifidobacteria show great diversity in the cell surface architecture which may influence the physicochemical properties of the bacterial cell and strain specific properties. The immunomodulatory role of bifidobacteria has been extensively studied, however studies on the immunoreactivity of their protein molecules are very limited. Here, we compared six different methods of protein isolation and purification and we report identification of immunogenic and immunoreactive protein of two human Bifidobacterium longum ssp. longum strains. We evaluated potential immunoreactive properties of proteins employing polyclonal sera obtained from germ free mouse, rabbit and human. The protein yield was isolation method-dependent and the reactivity of proteins detected by SDS-PAGE and Western blotting was heterogeneous and varied between different serum samples. The proteins with the highest immunoreactivity were isolated, purified and have them sequenced. Among the immunoreactive proteins we identified enolase, aspartokinase, pyruvate kinase, DnaK (B. longum ssp. longum CCM 7952) and sugar ABC transporter ATP-binding protein, phosphoglycerate kinase, peptidoglycan synthethase penicillin-binding protein 3, transaldolase, ribosomal proteins and glyceraldehyde 3-phosphate dehydrogenase (B. longum ssp. longum CCDM 372). PMID:27746766

Immunoreactive Proteins of Bifidobacterium longum ssp. longum CCM 7952 and Bifidobacterium longum ssp. longum CCDM 372 Identified by Gnotobiotic Mono-Colonized Mice Sera, Immune Rabbit Sera and Non-immune Human Sera.

PubMed

Górska, Sabina; Dylus, Ewa; Rudawska, Angelika; Brzozowska, Ewa; Srutkova, Dagmar; Schwarzer, Martin; Razim, Agnieszka; Kozakova, Hana; Gamian, Andrzej

2016-01-01

The Bifidobacteria show great diversity in the cell surface architecture which may influence the physicochemical properties of the bacterial cell and strain specific properties. The immunomodulatory role of bifidobacteria has been extensively studied, however studies on the immunoreactivity of their protein molecules are very limited. Here, we compared six different methods of protein isolation and purification and we report identification of immunogenic and immunoreactive protein of two human Bifidobacterium longum ssp. longum strains. We evaluated potential immunoreactive properties of proteins employing polyclonal sera obtained from germ free mouse, rabbit and human. The protein yield was isolation method-dependent and the reactivity of proteins detected by SDS-PAGE and Western blotting was heterogeneous and varied between different serum samples. The proteins with the highest immunoreactivity were isolated, purified and have them sequenced. Among the immunoreactive proteins we identified enolase, aspartokinase, pyruvate kinase, DnaK ( B. longum ssp. longum CCM 7952) and sugar ABC transporter ATP-binding protein, phosphoglycerate kinase, peptidoglycan synthethase penicillin-binding protein 3, transaldolase, ribosomal proteins and glyceraldehyde 3-phosphate dehydrogenase ( B. longum ssp. longum CCDM 372).

The histidine phosphocarrier protein, HPr, binds to the highly thermostable regulator of sigma D protein, Rsd, and its isolated helical fragments.

PubMed

Neira, José L; Hornos, Felipe; Cozza, Concetta; Cámara-Artigas, Ana; Abián, Olga; Velázquez-Campoy, Adrián

2018-02-01

The phosphotransferase system (PTS) controls the preferential use of sugars in bacteria and it is also involved in other processes, such as chemotaxis. It is formed by a protein cascade in which the first two proteins are general (namely, EI and HPr) and the others are sugar-specific permeases. The Rsd protein binds specifically to the RNA polymerase (RNAP) σ 70 factor. We first characterized the conformational stability of Escherichia coli Rsd. And second, we delineated the binding regions of Streptomyces coelicolor, HPr sc , and E. coli Rsd, by using fragments derived from each protein. To that end, we used several biophysical probes, namely, fluorescence, CD, NMR, ITC and BLI. Rsd had a free energy of unfolding of 15 kcal mol -1 at 25 °C, and a thermal denaturation midpoint of 103 °C at pH 6.5. The affinity between Rsd and HPr sc was 2 μM. Interestingly enough, the isolated helical-peptides, comprising the third (RsdH3) and fourth (RsdH4) Rsd helices, also interacted with HPr sc in a specific manner, and with affinities similar to that of the whole Rsd. Moreover, the isolated peptide of HPr sc , HPr 9-30 , comprising the active site, His15, also was bound to intact Rsd with similar affinity. Therefore, binding between Rsd and HPr sc was modulated by the two helices H3 and H4 of Rsd, and the regions around the active site of HPr sc . This implies that specific fragments of Rsd and HPr sc can be used to interfere with other protein-protein interactions (PPIs) of each other protein. Copyright © 2018 Elsevier Inc. All rights reserved.

A Mutation in the PP2C Phosphatase Gene in a Staphylococcus aureus USA300 Clinical Isolate with Reduced Susceptibility to Vancomycin and Daptomycin

PubMed Central

Passalacqua, Karla D.; Satola, Sarah W.; Crispell, Emily K.

2012-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (MIC of 4 to 8 μg/ml) are referred to as vancomycin-intermediate S. aureus (VISA). In this study, we characterized two isogenic USA300 S. aureus isolates collected sequentially from a single patient with endocarditis where the S. aureus isolate changed from being susceptible to vancomycin (VSSA) (1 μg/ml) to VISA (8 μg/ml). In addition, the VISA isolate lost beta-lactamase activity and showed increased resistance to daptomycin and linezolid. The two strains did not differ in growth rate, but the VISA isolate had a thickened cell wall and was less autolytic. Transcriptome sequencing (RNA-seq) analysis comparing the two isolates grown to late exponential phase showed significant differences in transcription of cell surface protein genes (spa, SBI [second immunoglobulin-binding protein of S. aureus], and fibrinogen-binding proteins), regulatory genes (agrBCA, RNAIII, sarT, and saeRS), and others. Using whole-genome shotgun resequencing, we identified 6 insertion/deletion mutations between the VSSA and VISA isolates. A protein phosphatase 2C (PP2C) family phosphatase had a 6-bp (nonframeshift) insertion mutation in a highly conserved metal binding domain. Complementation of the clinical VISA isolate with a wild-type copy of the PP2C gene reduced the vancomycin and daptomycin MICs and increased autolytic activity, suggesting that this gene contributed to the reduced vancomycin susceptibility phenotype acquired in vivo. Creation of de novo mutants from the VSSA strain resulted in different mutations, demonstrating that reduced susceptibility to vancomycin in USA300 strains can occur via multiple routes, highlighting the complex nature of the VISA phenotype. PMID:22850507

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

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

Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong

2016-01-08

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively boundmore » the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.« less

Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

PubMed Central

2013-01-01

The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein–protein interaction prediction and design methods. PMID:24250278

Size-dependent protein segregation at membrane interfaces

NASA Astrophysics Data System (ADS)

Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

2016-07-01

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

The presence of both bone sialoprotein-binding protein gene and collagen adhesin gene as a typical virulence trait of the major epidemic cluster in isolates from orthopedic implant infections.

PubMed

Campoccia, Davide; Speziale, Pietro; Ravaioli, Stefano; Cangini, Ilaria; Rindi, Simonetta; Pirini, Valter; Montanaro, Lucio; Arciola, Carla Renata

2009-12-01

Staphylococcus aureus is a major, highly clonal, pathogen causing implant infections. This study aimed at investigating the diverse distribution of bacterial adhesins in most prevalent S. aureus strain types causing orthopaedic implant infections. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes. Within the collection of isolates, automated ribotyping detected 98 distinct ribogroups. For many ribogroups, characteristic tandem genes arrangements could be identified. In the predominant S. aureus cluster, enlisting 27 isolates, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. This study suggests that specific adhesins may synergistically act in the onset of implant infections and that anti-adhesin strategies should be targeted to adhesins conjointly present.

Biopolymers: Protein and Nucleic Acids

DTIC Science & Technology

1987-09-15

characterized a 45 kDa "ZSB, de!•ignted S’SB1, most extensively. SSB1 was isolated on the basis of preferential binding to sin ie-stranded versus double...stranded DNA and was subsequently shown also to bind to RNA (9). Although SSB1 seems to bind without sequen-e specificity, it is interesting to note...that it copurifies through several a inity steps with the yeast poly (A) binding protein (11, Sach3 and Jong, unpublished observations). SSB1 stimulates

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

PubMed Central

Choe, Weonu; Durgannavar, Trishaladevi A.; Chung, Sang J.

2016-01-01

The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed. PMID:28774114

Trichoderma genes

DOEpatents

Foreman, Pamela [Los Altos, CA; Goedegebuur, Frits [Vlaardingen, NL; Van Solingen, Pieter [Naaldwijk, NL; Ward, Michael [San Francisco, CA

2012-06-19

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

Streptavidin mutants

DOEpatents

Sano, Takeshi; Cantor, Charles R.; Vajda, Sandor; Reznik, Gabriel O.; Smith, Cassandra L.; Pandori, Mark W.

2000-01-01

The present invention relates to streptavidin proteins and peptides having a altered physical properties such as an increased stability or increased or decreased affinity for binding biotin. The invention also relates to methods for the detection, identification, separation and isolation of targets using streptavidin proteins or peptides. Streptavidin with increased or reduced affinity allows for the use of the streptavidin-biotin coupling systems for detection and isolation systems wherein it is necessary to remove of one or the other of the binding partners. Such systems are useful for the purification of functional proteins and viable cells. The invention also relates to nucleic acids which encode these streptavidin proteins and peptides and to recombinant cells such as bacteria, yeast and mammalian cells which contain these nucleic acids.

Isolation and characterization of a cDNA encoding a membrane bound acyl-CoA binding protein from Agave americana L. epidermis.

PubMed

Guerrero, Consuelo; Martín-Rufián, M; Reina, José J; Heredia, Antonio

2006-01-01

A cDNA encoding an acyl-CoA binding protein (ACBP) homologue has been cloned from a cDNA library made from mRNA isolated from epidermis of young leaves of Agave americana L. The derived amino acid sequence reveals a protein corresponding to the membrane-associated form of ACBPs only previously described in Arabidopsis and rice. Northern blot analysis showed that the A. americana ACBP gene is mainly expressed in the epidermis of mature zone of the leaves. The epidermis of A. americana leaves have a well developed cuticle with the highest amounts of the cuticular components waxes, cutin and cutan suggesting a potential role of the protein in cuticle formation.

Echinococcus granulosus fatty acid binding proteins subcellular localization.

PubMed

Alvite, Gabriela; Esteves, Adriana

2016-05-01

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Response of Staphylococcus aureus isolates from bovine mastitis to exogenous iron sources.

PubMed

Diarra, M S; Petitclerc, D; Lacasse, P

2002-09-01

Staphylococcus aureus can survive in conditions of extremely low iron concentration. The ability of S. aureus to use two exogenous hydroxamate types of siderophores (desferrioxamine and ferrichrome) and four iron-containing proteins found in cattle (hemin, hemoglobin, ferritin, and lactoferrin) were tested on 16 reference and clinical isolates. For all strains tested, ferrichrome and desferrioxamine showed strong growth-promoting activities in a disk diffusion assay and in liquid medium. The heme proteins hemin and hemoglobin were also found to support growth in culture media lacking other iron sources, while lactoferrin failed to do so. On media containing the iron chelator dipyridyl, ferritin induced a growth inhibition effect that was further enhanced in the presence of lactoferrin in seven of the 13 tested strains. Staphylococcus aureus was able to bind hemin and the level of binding activity was not increased after growth in iron-rich or -poor media. Dot-blot competition tests showed that biotin-labeled lactoferrin binds to S. aureus, and this binding can be inhibited by unlabeled lactoferrin. Expression of lactoferrin-binding activity was independent of the level of iron in the medium and the iron saturation status of lactoferrin. For each strain tested, ligand blots showed lactoferrin-binding proteins of molecular weights ranging from 32 to 92 kDa. Possible functions of these lactoferrin-binding proteins could not be related to iron acquisition mechanism in S. aureus.

Prevalence of factor H-binding protein variants and NadA among meningococcal group B isolates from the United States: implications for the development of a multicomponent group B vaccine.

PubMed

Beernink, Peter T; Welsch, Jo Anne; Harrison, Lee H; Leipus, Arunas; Kaplan, Sheldon L; Granoff, Dan M

2007-05-15

Two promising recombinant meningococcal protein vaccines are in development. One contains factor H-binding protein (fHBP) variants (v.) 1 and 2, whereas the other contains v.1 and 4 other antigens discovered by genome mining (5 component [5C]). Antibodies against fHBP are bactericidal against strains within a variant group. There are limited data on the prevalence of strains expressing different fHBP variants in the United States. A total of 143 group B isolates from patients hospitalized in the United States were tested for fHBP variant by quantitative polymerase chain reaction, for reactivity with 6 anti-fHBP monoclonal antibodies (MAb) by dot immunoblotting, and for susceptibility to bactericidal activity of mouse antisera. fHBP v.1 isolates predominated in California (83%), whereas isolates expressing v.1 (53%) or v.2 (42%) were common in 9 other states. Isolates representative of 5 anti-fHBP MAb-binding phenotypes (70% of isolates) were highly susceptible to anti-fHBP v.1 or v.2 bactericidal activity, whereas 3 phenotypes were approximately 50% susceptible. Collectively, antibodies against the fHBP v.1 and v.2 vaccine and the 5C vaccine killed 76% and 83% of isolates, respectively. Susceptibility to bactericidal activity can be predicted, in part, on the basis of fHBP phenotypes. Both vaccines have the potential to prevent most group B disease in the United States.

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

NASA Technical Reports Server (NTRS)

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

1996-01-01

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

Recombinant soluble adenovirus receptor

DOEpatents

Freimuth, Paul I.

2002-01-01

Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

Scaffold diversification enhances effectiveness of a superlibrary of hyperthermophilic proteins.

PubMed

Hussain, Mahmud; Gera, Nimish; Hill, Andrew B; Rao, Balaji M

2013-01-18

The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in biotechnology and medicine. Typically, binders are isolated from a combinatorial library generated by mutating a single scaffold protein. In contrast, here we generated a "superlibrary" or "library-of-libraries" of 4 × 10(8) protein variants by mutagenesis of seven different hyperthermophilic proteins; six of the seven proteins have not been used as scaffolds prior to this study. Binding proteins for five different model targets were successfully isolated from this library. Binders obtained were derived from five out of the seven scaffolds. Strikingly, binders from this modestly sized superlibrary have affinities comparable or higher than those obtained from a library with 1000-fold higher sequence diversity but derived from a single stable scaffold. Thus scaffold diversification, i.e., randomization of multiple different scaffolds, is a powerful alternate strategy for combinatorial library construction.

Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

PubMed Central

Hansen, Randi Westh; Wang, Xiaole; Golab, Agnieszka; Bornert, Olivier; Oswald, Christine; Wagner, Renaud; Martinez, Karen Laurence

2016-01-01

Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. PMID:27035823

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions

PubMed Central

Lee, Wonbae; Gillies, John P.; Jose, Davis; Israels, Brett A.; von Hippel, Peter H.; Marcus, Andrew H.

2016-01-01

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed. PMID:27694621

Actinomyces naeslundii Displays Variant fimP and fimA Fimbrial Subunit Genes Corresponding to Different Types of Acidic Proline-Rich Protein and β-Linked Galactosamine Binding Specificity

PubMed Central

Hallberg, K.; Holm, C.; Öhman, U.; Strömberg, N.

1998-01-01

Actinomyces naeslundii genospecies 1 and 2 bind to acidic proline-rich proteins (APRPs) and statherin via type 1 fimbriae and to β-linked galactosamine (GalNAcβ) structures via type 2 fimbriae. In addition, A. naeslundii displays two types of binding specificity for both APRPs-statherin and GalNAcβ, while Actinomyces odontolyticus binds to unknown structures. To study the molecular basis for these binding specificities, DNA fragments spanning the entire or central portions of fimP (type 1) and fimA (type 2) fimbrial subunit genes were amplified by PCR from strains of genospecies 1 and 2 and hybridized with DNA from two independent collections of oral Actinomyces isolates. Isolates of genospecies 1 and 2 and A. odontolyticus, but no other Actinomyces species, were positive for hybridization with fimP and fimA full-length probes irrespective of binding to APRPs and statherin, GalNAcβ, or unknown structures. Isolates of genospecies 1 and 2, with deviating patterns of GalNAcβ1-3Galα-O-ethyl-inhibitable coaggregation with Streptococcus oralis Ss34 and MPB1, were distinguished by a fimA central probe from genospecies 1 and 2, respectively. Furthermore, isolates of genospecies 1 and 2 displaying preferential binding to APRPs over statherin were positive with a fimP central probe, while a genospecies 2 strain with the opposite binding preference was not. The sequences of fimP and fimA central gene segments were highly conserved among isolates with the same, but diversified between those with a variant, binding specificity. In conclusion, A. naeslundii exhibits variant fimP and fimA genes corresponding to diverse APRP and GalNAcβ specificities, respectively, while A. odontolyticus has a genetically related but distinct adhesin binding specificity. PMID:9712794

CIP1 polypeptides and their uses

DOEpatents

Foreman, Pamela [Los Altos, CA; Van Solingen, Pieter [Naaldwijk, NL; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA

2011-04-12

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae

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

Yamada, Mototsugu, E-mail: mototsugu-yamada@meiji.co.jp; Watanabe, Takashi; Baba, Nobuyoshi

The selenomethionyl-substituted transpeptidase domain of penicillin-binding protein (PBP) 2B from S. pneumoniae was isolated from a limited proteolysis digest of the soluble form of recombinant PBP 2B and then crystallized. MAD data were collected to 2.4 Å resolution. Penicillin-binding protein (PBP) 2B from Streptococcus pneumoniae catalyzes the cross-linking of peptidoglycan precursors that occurs during bacterial cell-wall biosynthesis. A selenomethionyl (SeMet) substituted PBP 2B transpeptidase domain was isolated from a limited proteolysis digest of a soluble form of recombinant PBP 2B and then crystallized. The crystals belonged to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 86.39,more » c = 143.27 Å. Diffraction data were collected to 2.4 Å resolution using the BL32B2 beamline at SPring-8. The asymmetric unit contains one protein molecule and 63.7% solvent.« less

Negative inotropic effect of carbachol and interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein in mouse isolated atrium--a novel methodological trial.

PubMed

Okada, Muneyoshi; Noma, Chihiro; Yamawaki, Hideyuki; Hara, Yukio

2013-01-01

Interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein was examined by an immunoprecipitation-Western blotting system in mouse isolated atrium. The carbachol-induced negative inotropic action in indomethacin-pretreated mouse atrium was significantly inhibited by a K.ACh channel blocker, tertiapin or atropine. Kir3.1 K.ACh channel (Kir3.1) was immunoprecipitated with a mouse anti-Kir3.1 antibody. Coprecipitating Gβ with Kir3.1, detected by Western blotting, was significantly augmented by carbachol. Atropine, but not tertiapin, significantly inhibited the carbachol-induced coprecipitating Gβ with Kir3.1. The data indicate that immunoprecipitation with Kir3.1 and Western blotting of Gβ system is a useful method for assessing interaction between K.ACh channel and GTP binding protein in mouse atrium.

Imperfect duplicate insertions type of mutations in plasmepsin V modulates binding properties of PEXEL motifs of export proteins in Indian Plasmodium vivax.

PubMed

Rawat, Manmeet; Vijay, Sonam; Gupta, Yash; Tiwari, Pramod Kumar; Sharma, Arun

2013-01-01

Plasmepsin V (PM-V) have functionally conserved orthologues across the Plasmodium genus who's binding and antigenic processing at the PEXEL motifs for export about 200-300 essential proteins is important for the virulence and viability of the causative Plasmodium species. This study was undertaken to determine P. vivax plasmepsin V Ind (PvPM-V-Ind) PEXEL motif export pathway for pathogenicity-related proteins/antigens export thereby altering plasmodium exportome during erythrocytic stages. We identify and characterize Plasmodium vivax plasmepsin-V-Ind (mutant) gene by cloning, sequence analysis, in silico bioinformatic protocols and structural modeling predictions based on docking studies on binding capacity with PEXEL motifs processing in terms of binding and accessibility of export proteins. Cloning and sequence analysis for genetic diversity demonstrates PvPM-V-Ind (mutant) gene is highly conserved among all isolates from different geographical regions of India. Imperfect duplicate insertion types of mutations (SVSE from 246-249 AA and SLSE from 266-269 AA) were identified among all Indian isolates in comparison to P.vivax Sal-1 (PvPM-V-Sal 1) isolate. In silico bioinformatics interaction studies of PEXEL peptide and active enzyme reveal that PvPM-V-Ind (mutant) is only active in endoplasmic reticulum lumen and membrane embedding is essential for activation of plasmepsin V. Structural modeling predictions based on docking studies with PEXEL motif show significant variation in substrate protein binding of these imperfect mutations with data mined PEXEL sequences. The predicted variation in the docking score and interacting amino acids of PvPM-V-Ind (mutant) proteins with PEXEL and lopinavir suggests a modulation in the activity of PvPM-V in terms of binding and accessibility at these sites. Our functional modeled validation of PvPM-V-Ind (mutant) imperfect duplicate insertions with data mined PEXEL sequences leading to altered binding and substrate accessibility of the enzyme makes it a plausible target to investigate export mechanisms for in silico virtual screening and novel pharmacophore designing.

Imperfect Duplicate Insertions Type of Mutations in Plasmepsin V Modulates Binding Properties of PEXEL Motifs of Export Proteins in Indian Plasmodium vivax

PubMed Central

Rawat, Manmeet; Vijay, Sonam; Gupta, Yash; Tiwari, Pramod Kumar; Sharma, Arun

2013-01-01

Introduction Plasmepsin V (PM-V) have functionally conserved orthologues across the Plasmodium genus who's binding and antigenic processing at the PEXEL motifs for export about 200–300 essential proteins is important for the virulence and viability of the causative Plasmodium species. This study was undertaken to determine P. vivax plasmepsin V Ind (PvPM-V-Ind) PEXEL motif export pathway for pathogenicity-related proteins/antigens export thereby altering plasmodium exportome during erythrocytic stages. Method We identify and characterize Plasmodium vivax plasmepsin-V-Ind (mutant) gene by cloning, sequence analysis, in silico bioinformatic protocols and structural modeling predictions based on docking studies on binding capacity with PEXEL motifs processing in terms of binding and accessibility of export proteins. Results Cloning and sequence analysis for genetic diversity demonstrates PvPM-V-Ind (mutant) gene is highly conserved among all isolates from different geographical regions of India. Imperfect duplicate insertion types of mutations (SVSE from 246–249 AA and SLSE from 266–269 AA) were identified among all Indian isolates in comparison to P.vivax Sal-1 (PvPM-V-Sal 1) isolate. In silico bioinformatics interaction studies of PEXEL peptide and active enzyme reveal that PvPM-V-Ind (mutant) is only active in endoplasmic reticulum lumen and membrane embedding is essential for activation of plasmepsin V. Structural modeling predictions based on docking studies with PEXEL motif show significant variation in substrate protein binding of these imperfect mutations with data mined PEXEL sequences. The predicted variation in the docking score and interacting amino acids of PvPM-V-Ind (mutant) proteins with PEXEL and lopinavir suggests a modulation in the activity of PvPM-V in terms of binding and accessibility at these sites. Conclusion/Significance Our functional modeled validation of PvPM-V-Ind (mutant) imperfect duplicate insertions with data mined PEXEL sequences leading to altered binding and substrate accessibility of the enzyme makes it a plausible target to investigate export mechanisms for in silico virtual screening and novel pharmacophore designing. PMID:23555891

Biochemical and physicochemical analysis of fish protein isolate recovered from red snapper (Lutjanus sp.) by-product using isoelectric solubilization/precipitation method

NASA Astrophysics Data System (ADS)

Pramono, H.; Pujiastuti, D. Y.; Sahidu, A. M.

2018-04-01

The effect of acid- and alkali-process on biochemical and physicochemical characteristics of fish protein isolate from red snapper (Lutjanus sp) by-product was evaluated. Protein recovered by alkali process (16.79%) was higher compared to acid process (13.75%). Reduction of lipid content and total volatile basic nitrogen (TVB-N) exhibited in both treatments indicated both process improved fish protein isolate recovered from red snapper by-product. In addition, the increasing of water holding capacity and oil binding capacity were observed. However, high peroxide value of fish protein isolate was showed in both treatment. This finding indicated that acid and alkali process can be used as a useful method to recover proteins from red snapper by-product. Alkali process gave a protein isolate with better overall quality compared to acid process.

Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates.

PubMed

Arnold, A R; Burnham, C-A D; Ford, B A; Lawhon, S D; McAllister, S K; Lonsway, D; Albrecht, V; Jerris, R C; Rasheed, J K; Limbago, B; Burd, E M; Westblade, L F

2016-03-01

The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Characterization of a gene coding for a type IIo bacterial IgG-binding protein.

PubMed

Boyle, M D; Weber-Heynemann, J; Raeder, R; Podbielski, A

1995-06-01

Two antigenic classes of non-immune IgG-binding proteins can be expressed by group A streptococci. One antigenic group of proteins is recognized by an antibody prepared against the product of a cloned fcrA gene (anti-FcRA). In this study, the immunogen used to prepare the antibody that defines the second antigenic class was shown to be the product of the emm-like (emmL) gene of M serotype 55 group A isolate, A928. The emmL55 gene expressed in E. coli produced an M(r) approximately 58,000 molecule which bound human IgG1, IgG2, IgG3 and IgG4, as well as horse, rabbit and pig IgG in a non-immune fashion. These properties are characteristic of the previously described type IIo IgG-binding protein isolated from this strain. In addition, the recombinant protein was reactive with human serum albumin and fibrinogen. The emmL 55 gene sequence was analysed and found to have the organization and sequence characteristics of a typical class I emm-like gene.

SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G

PubMed Central

Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P.; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus

2017-01-01

The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis. PMID:28401063

Possible Insecticidal Mechanisms Mediated by Immune-Response-Related Cry-Binding Proteins in the Midgut Juice of Plutella xylostella and Spodoptera exigua.

PubMed

Lu, Keyu; Gu, Yuqing; Liu, Xiaoping; Lin, Yi; Yu, Xiao-Qiang

2017-03-15

Cry toxins are insecticidal toxin proteins produced by a spore-forming Gram-positive bacterium Bacillus thuringiensis. Interactions between the Cry toxins and the receptors from midgut brush border membrane vesicles (BBMVs), such as cadherin, alkaline phosphatase, and aminopeptidase, are key steps for the specificity and insecticidal activity of Cry proteins. However, little is known about the midgut juice proteins that may interfere with Cry binding to the receptors. To validate the hypothesis that there exist Cry-binding proteins that can interfere with the insecticidal process of Cry toxins, we applied Cry1Ab1-coupled Sepharose beads to isolate Cry-binding proteins form midgut juice of Plutella xylostella and Spodoptera exigua. Trypsin-like serine proteases and Dorsal were found to be Cry1Ab1-binding proteins in the midgut juice of P. xylostella. Peroxidase-C (POX-C) was found to be the Cry1Ab1-binding protein in the midgut juice of S. exigua. We proposed possible insecticidal mechanisms of Cry1Ab1 mediated by the two immune-related proteins: Dorsal and POX-C. Our results suggested that there exist, in the midgut juice, Cry-binding proteins, which are different from BBMV-specific receptors.

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

PubMed

Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

1997-03-01

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

Surface energetics and protein-protein interactions: analysis and mechanistic implications

PubMed Central

Peri, Claudio; Morra, Giulia; Colombo, Giorgio

2016-01-01

Understanding protein-protein interactions (PPI) at the molecular level is a fundamental task in the design of new drugs, the prediction of protein function and the clarification of the mechanisms of (dis)regulation of biochemical pathways. In this study, we use a novel computational approach to investigate the energetics of aminoacid networks located on the surface of proteins, isolated and in complex with their respective partners. Interestingly, the analysis of individual proteins identifies patches of surface residues that, when mapped on the structure of their respective complexes, reveal regions of residue-pair couplings that extend across the binding interfaces, forming continuous motifs. An enhanced effect is visible across the proteins of the dataset forming larger quaternary assemblies. The method indicates the presence of energetic signatures in the isolated proteins that are retained in the bound form, which we hypothesize to determine binding orientation upon complex formation. We propose our method, BLUEPRINT, as a complement to different approaches ranging from the ab-initio characterization of PPIs, to protein-protein docking algorithms, for the physico-chemical and functional investigation of protein-protein interactions. PMID:27050828

Isolation and characterization of an RNA aptamer for the HPV-16 E7 oncoprotein.

PubMed

Toscano-Garibay, Julia D; Benítez-Hess, María L; Alvarez-Salas, Luis M

2011-02-01

Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer. Copyright © 2011 IMSS. Published by Elsevier Inc. All rights reserved.

Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.).

PubMed

Park, S H; Strobel, G A

1994-01-05

Maculosin (the diketopiperazine, cyclo (L-Pro-L-Tyr)) is a host specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa L.). Receptors for this phytotoxin have been isolated from spotted knapweed. Knapweed leaves possess most of the maculosin-binding activity in the cytosolic fraction. However, activity was also observed in the whole membrane fraction of the leaf. The binding component of the cytosolic fraction was identified as a protein(s) because of its heat-lability and sensitivity to proteases. A 16-fold purification of a toxin-binding protein was carried out by ammonium sulfate fractionation, and Sephadex G-200, and maculosin-affinity column chromatography. The affinity column was prepared with epoxy activated Sepharose 6B to which the phenolic group of maculosin was attached. The receptor was estimated to contain more than one binding protein by native and SDS-PAGE. At least one of the maculosin-binding proteins was identified as ribulose-1,5-biphosphate carboxylase (RuBPcase).

Annexin II is associated with mRNAs which may constitute a distinct subpopulation.

PubMed Central

Vedeler, A; Hollås, H

2000-01-01

Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of (32)P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)(+)-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs. PMID:10839987

Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks

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

Mans, Ben J.; Ribeiro, Jose M.C.; Andersen, John F.

2008-08-19

Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to themore » lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.« less

The isolated, twenty-three-residue-long, N-terminal region of the glutamine synthetase inactivating factor binds to its target.

PubMed

Neira, José L; Florencio, Francisco J; Muro-Pastor, M Isabel

2017-09-01

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS type I is regulated by protein-protein interactions with a 65-residue-long protein (IF7). IF7 binds initially to GS through residues at its N terminus. In this work, we studied the conformational preferences of the N-terminal region of IF7 (IF7pep, residues Ala7-Ala29), its binding to GS and its functional properties. Isolated IF7pep populated a nascent helix in aqueous solution. IF7pep was bound to GS with an affinity constant of 0.4μM, and a 1:1 stoichiometry. IF7pep did not inactivate GS, suggesting that there were other IF7 regions important to carry out the inactivating function. Binding of IF7pep to GS was electrostatically-driven and it did not follow a kinetic two-state model. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly selective isolation and purification of heme proteins in biological samples using multifunctional magnetic nanospheres.

PubMed

Liu, Yating; Li, Yan; Wei, Yun

2014-12-01

Magnetic particles with suitable surface modification are capable of binding proteins selectively, and magnetic separations have advantages of rapidity, convenience, and high selectivity. In this paper, new magnetic nanoparticles modified with imidazolium ionic liquid (Fe3O4 @SiO2 @ILs) were successfully fabricated. N-Methylimidazolium was immobilized onto silica-coated magnetic nanoparticles via γ-chloropropyl modification as a magnetic nanoadsorbent for heme protein separation. The particle size was about 90 nm without significant aggregation during the preparation process. Hemoglobin as one of heme proteins used in this experiment was compared with other nonheme proteins. It has been found that the magnetic nanoparticles can be used for more rapid, efficient, and specific adsorption of hemoglobin with a binding capacity as high as 5.78 mg/mg. In comparison with other adsorption materials of proteins in the previous reports, Fe3 O4 @SiO2 @ILs magnetic nanoparticles exhibit the excellent performance in isolation of heme proteins with higher binding capacity and selectivity. In addition, a short separation time makes the functionalized nanoparticles suitable for purifying unstable proteins, as well as having other potential applications in a variety of biomedical fields. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrostatic interaction of positive charges on the surface of Psb31 with photosystem II in the diatom Chaetoceros gracilis.

PubMed

Nagao, Ryo; Suzuki, Takehiro; Okumura, Akinori; Kihira, Tomohiro; Toda, Ayaka; Dohmae, Naoshi; Nakazato, Katsuyoshi; Tomo, Tatsuya

2017-09-01

Psb31, a novel extrinsic protein found in diatom photosystem II (PSII), directly binds to PSII core subunits, independent of the other extrinsic proteins, and functions to maintain optimum oxygen evolution. However, how Psb31 electrostatically interacts with PSII intrinsic proteins remains to be clarified. In this study, we examined electrostatic interaction of Psb31 with PSII complexes isolated from the diatom Chaetoceros gracilis. Positive or negative charges of isolated Psb31 proteins were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME), respectively, resulting in formation of uncharged groups. NSP-modified Psb31 did not bind to PSII with a concomitant increase in NSP concentration, whereas GME-modified Psb31 clearly bound to PSII with retention of oxygen-evolving activity, indicating that positive charges of Lys residues and the N-terminus on the surface of Psb31 are involved in electrostatic interactions with PSII intrinsic proteins. Mass spectrometry analysis of NSP-modified Psb31 and sequence comparisons of Psb31 from C. gracilis with other chromophyte algae led to identification of three Lys residues as possible binding sites to PSII. Based on these findings, together with our previous cross-linking study in diatom PSII and a red algal PSII structure, we discuss binding properties of Psb31 with PSII core proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Myostatin inhibits porcine intramuscular preadipocyte differentiation in vitro.

PubMed

Sun, W X; Dodson, M V; Jiang, Z H; Yu, S G; Chu, W W; Chen, J

2016-04-01

This study assessed the effect of myostatin on adipogenesis by porcine intramuscular preadipocytes. Intramuscular preadipocytes were isolated from the longissimus dorsi muscle of newborn pigs. Myostatin inhibited intramuscular preadipocyte differentiation in a dose-dependent manner. Myostatin treatment during preadipocyte differentiation significantly (P < 0.05) inhibited the expression of the adipogenic marker genes CCAAT/enhancer-binding protein β, CCAAT/enhancer-binding protein α, peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein-1c, fatty acid-binding protein, and adiponectin. Myostatin also significantly (P < 0.05) reduced the release of glycerol and decreased both adipose triglyceride lipase and hormone-sensitive lipase expression in intramuscular adipocytes. Our study suggests that myostatin acts as an extrinsic regulatory factor in regulating intramuscular adipogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of a maize nucleic acid-binding protein (NBP) belonging to a family of nuclear-encoded chloroplast proteins.

PubMed Central

Cook, W B; Walker, J C

1992-01-01

A cDNA encoding a nuclear-encoded chloroplast nucleic acid-binding protein (NBP) has been isolated from maize. Identified as an in vitro DNA-binding activity, NBP belongs to a family of nuclear-encoded chloroplast proteins which share a common domain structure and are thought to be involved in posttranscriptional regulation of chloroplast gene expression. NBP contains an N-terminal chloroplast transit peptide, a highly acidic domain and a pair of ribonucleoprotein consensus sequence domains. NBP is expressed in a light-dependent, organ-specific manner which is consistent with its involvement in chloroplast biogenesis. The relationship of NBP to the other members of this protein family and their possible regulatory functions are discussed. Images PMID:1346929

Soy protein isolate and estradiol differ in their effects on the mammary gland of weanling male and female rats

USDA-ARS?s Scientific Manuscript database

Isoflavones are phytochemical components of soy diets that bind weakly to estrogen receptors (ERs). To study potential estrogen-like actions of soy in the mammary gland, we fed weanling male and female Sprague-Dawley rats a casein diet from PND21 to PND33, the same diet substituting soy protein isol...

Isolation and characterizations of oxalate-binding proteins in the kidney

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

Roop-ngam, Piyachat; Chaiyarit, Sakdithep; Pongsakul, Nutkridta

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

Effect of resveratrol or ascorbic acid on the stability of α-tocopherol in O/W emulsions stabilized by whey protein isolate: Simultaneous encapsulation of the vitamin and the protective antioxidant.

PubMed

Wang, Lei; Gao, Yahui; Li, Juan; Subirade, Muriel; Song, Yuanda; Liang, Li

2016-04-01

Food proteins have been widely used as carrier materials due to their multiple functional properties. Hydrophobic bioactives are generally dissolved in the oil phase of O/W emulsions. Ligand-binding properties provide the possibility of binding bioactives to the protein membrane of oil droplets. In this study, the influence of whey protein isolate (WPI) concentration and amphiphilic resveratrol or hydrophilic ascorbic acid on the decomposition of α-tocopherol in the oil phase of WPI emulsions is considered. Impact of ascorbic acid, in the continuous phase, on the decomposition depended on the vitamin concentration. Resveratrol partitioned into the oil-water interface and the cis-isomer contributed most of the protective effect of this polyphenol. About 94% of α-tocopherol and 50% of resveratrol were found in the oil droplets stabilized by 0.01% WPI. These results suggest the feasibility of using the emulsifying and ligand-binding properties of WPI to produce carriers for simultaneous encapsulation of bioactives with different physicochemical properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

Definition of IgG- and albumin-binding regions of streptococcal protein G.

PubMed

Akerström, B; Nielsen, E; Björck, L

1987-10-05

Protein G, the immunoglobin G-binding surface protein of group C and G streptococci, also binds serum albumin. The albumin-binding site on protein G is distinct from the immunoglobulin G-binding site. By mild acid hydrolysis of the papain-liberated protein G fragment (35 kDa), a 28-kDa fragment was produced which retained full immunoglobulin G-binding activity (determined by Scatchard plotting) but had lost all albumin-binding capacity. A protein G (65 kDa), isolated after cloning and expression of the protein G gene in Escherichia coli, had comparable affinity to immunoglobulin G (5-10 X 10(10)M-1), but much higher affinity to albumin than the 35- and 28-kDa protein G fragments (31, 2.6, and 0 X 10(9)M-1, respectively). The amino-terminal amino acid sequences of the 65-, 35-, and 28-kDa fragments allowed us to exactly locate the three fragments in an overall sequence map of protein G, based on the partial gene sequences published by Guss et al. (Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.-K., Jörnvall, H., Flock, J.-I., and Lindberg, M. (1986) EMBO J. 5, 1567-1575) and Fahnestock et al. (Fahnestock, S. R., Alexander, P., Nagle, J., and Filpula, D. (1986) J. Bacteriol. 167, 870-880). In this map could then be deduced the location of three homologous albumin-binding regions and three homologous immunoglobulin G-binding regions.

A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences

PubMed Central

Nagano, Yukio; Furuhashi, Hirofumi; Inaba, Takehito; Sasaki, Yukiko

2001-01-01

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x2-H-x11-C-x2-C-x(4–5)-C-x2-C-x(3–7)-H-x2-H and C-x2-C-x(10–11)-C-x3-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with 65ZnCl2 showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression. PMID:11600698

Isolation and Characterization of Two Proteins from Moraxella catarrhalis That Bear a Common Epitope

PubMed Central

McMichael, John C.; Fiske, Michael J.; Fredenburg, Ross A.; Chakravarti, Deb N.; VanDerMeid, Karl R.; Barniak, Vicki; Caplan, Jeffrey; Bortell, Eric; Baker, Steven; Arumugham, Rasappa; Chen, Dexiang

1998-01-01

The UspA1 and UspA2 proteins of Moraxella catarrhalis are potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates. PMID:9712790

Vanadium-Binding Ability of Nucleoside Diphosphate Kinase from the Vanadium-Rich Fan Worm, Pseudopotamilla occelata.

PubMed

Yamaguchi, Nobuo; Yoshinaga, Masafumi; Kamino, Kei; Ueki, Tatsuya

2016-06-01

Polychaete fan worms and ascidians accumulate high levels of vanadium ions. Several vanadiumbinding proteins, known as vanabins, have been found in ascidians. However, no vanadium-binding factors have been isolated from the fan worm. In the present study, we sought to identify vanadiumbinding proteins in the branchial crown of the fan worm using immobilized metal ion affinity chromatography. A nucleoside diphosphate kinase (NDK) homolog was isolated and determined to be a vanadium-binding protein. Kinase activity of the NDK homologue, PoNDK, was suppressed by the addition of V(IV), but was unaffected by V(V). The effect of V(IV) on PoNDK precedes its activation by Mg(II). This is the first report to describe the relationship between NDK and V(IV). PoNDK is located in the epidermis of the branchial crown, and its distribution is very similar to that of vanadium. These results suggest that PoNDK is associated with vanadium accumulation and metabolism in P. occelata.

Characterization of a Complement-Binding Protein, DRS, from Strains of Streptococcus pyogenes Containing the emm12 and emm55 Genes

PubMed Central

Binks, Michael; Sriprakash, K. S.

2004-01-01

An extracellular protein of Streptococcus pyogenes, streptococcal inhibitor of complement (SIC), and its variant, called DRS (distantly related to SIC), are expressed by some S. pyogenes strains. SIC from type 1 (M1) isolates of S. pyogenes interferes with complement-mediated cell lysis, reportedly via its interaction with complement proteins. In this study we demonstrate that S. pyogenes strains carrying emm12 and emm55 (the genes for the M12 and M55 proteins, respectively) express and secrete DRS. This protein, like SIC, binds to the C6 and C7 complement proteins, and competition enzyme-linked immunosorbent assay experiments demonstrate that DRS competes with SIC for C6 and C7 binding. Similarly, SIC competes with DRS for binding to the complement proteins. Despite this, the recombinant DRS preparation showed no significant effect on complement function, as determined by lysis of sensitized sheep erythrocytes. Furthermore, the presence of DRS is not inhibitory to SIC activity. PMID:15213143

Characterization of a complement-binding protein, DRS, from strains of Streptococcus pyogenes containing the emm12 and emm55 genes.

PubMed

Binks, Michael; Sriprakash, K S

2004-07-01

An extracellular protein of Streptococcus pyogenes, streptococcal inhibitor of complement (SIC), and its variant, called DRS (distantly related to SIC), are expressed by some S. pyogenes strains. SIC from type 1 (M1) isolates of S. pyogenes interferes with complement-mediated cell lysis, reportedly via its interaction with complement proteins. In this study we demonstrate that S. pyogenes strains carrying emm12 and emm55 (the genes for the M12 and M55 proteins, respectively) express and secrete DRS. This protein, like SIC, binds to the C6 and C7 complement proteins, and competition enzyme-linked immunosorbent assay experiments demonstrate that DRS competes with SIC for C6 and C7 binding. Similarly, SIC competes with DRS for binding to the complement proteins. Despite this, the recombinant DRS preparation showed no significant effect on complement function, as determined by lysis of sensitized sheep erythrocytes. Furthermore, the presence of DRS is not inhibitory to SIC activity.

Peroxisome proliferator-binding protein: identification and partial characterization of nafenopin-, clofibric acid-, and ciprofibrate-binding proteins from rat liver.

PubMed Central

Lalwani, N D; Alvares, K; Reddy, M K; Reddy, M N; Parikh, I; Reddy, J K

1987-01-01

Peroxisome proliferators (PP) induce a highly predictable pleiotropic response in rat and mouse liver that is characterized by hepatomegaly, increase in peroxisome number in hepatocytes, and induction of certain peroxisomal enzymes. The PP-binding protein (PPbP) was purified from rat liver cytosol by a two-step procedure involving affinity chromatography and ion-exchange chromatography. Three PP, nafenopin and its structural analogs clofibric acid and ciprofibrate, were used as affinity ligands and eluting agents. This procedure yields a major protein with an apparent Mr of 70,000 on NaDodSO4/PAGE in the presence of reducing agent and Mr 140,000 (Mr 140,000-160,000) on gel filtration and polyacrylamide gradient gel electrophoresis under nondenaturing conditions, indicating that the active protein is a dimer. This protein has an acidic pI of 4.2 under nondenaturing conditions, which rises to 5.6 under denaturing conditions. The isolation of the same Mr 70,000 protein with three different, but structurally related, agents as affinity ligands and the immunological identity of the isolated proteins constitute strong evidence that this protein is the PPbP capable of recognizing PP that are structurally related to clofibrate. The PPbP probably plays an important role in the regulation of PP-induced pleiotropic response. Images PMID:3474650

Dieckol, a phlorotannin isolated from a brown seaweed, Ecklonia cava, inhibits adipogenesis through AMP-activated protein kinase (AMPK) activation in 3T3-L1 preadipocytes.

PubMed

Ko, Seok-Chun; Lee, Myoungsook; Lee, Ji-Hyeok; Lee, Seung-Hong; Lim, Yunsook; Jeon, You-Jin

2013-11-01

In this study, we assessed the potential inhibitory effect of 5 species of brown seaweeds on adipogenesis the differentiation of 3T3-L1 preadipocytes into mature adipocytes by measuring Oil-Red O staining. The Ecklonia cava extract tested herein evidenced profound adipogenesis inhibitory effect, compared to that exhibited by the other four brown seaweed extracts. Thus, E. cava was selected for isolation of active compounds and finally the three polyphenol compounds of phlorotannins were obtained and their inhibitory effect on adipogenesis was observed. Among the phlorotannins, dieckol exhibited greatest potential adipogenesis inhibition and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding proteins (C/EBPα), sterol regulatory element-binding protein 1 (SREBP1) and fatty acid binding protein 4 (FABP4) in a dose-dependent manner. The specific mechanism mediating the effects of dieckol was confirmed by AMP-activated protein kinase (AMPK) activation. These results demonstrate inhibitory effect of dieckol compound on adipogenesis through the activation of the AMPK signal pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

Targeting of cytosolic mRNA to mitochondria: naked RNA can bind to the mitochondrial surface.

PubMed

Michaud, Morgane; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie

2014-05-01

Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Phage selection of peptide "microantibodies".

PubMed

Fujiwara, Daisuke; Fujii, Ikuo

2013-01-01

A bioactive peptide capable of inhibiting protein-protein interactions has the potential to be a molecular tool for biological studies and a therapeutic by disrupting aberrant interactions involved in diseases. We have developed combinatorial libraries of peptides with helix-loop-helix structure, from which the isolated peptides have the constrained structure to reduce entropy costs in binding, resulting in high binding affinities for target molecules. Previously, we designed a de novo peptide of helix-loop-helix structure that we termed a "microantibody." Using the microantibody as a library scaffold, we have constructed a phage-display library to successfully isolate molecular-targeting peptides against a cytokine receptor (granulocyte colony-stimulating factor receptor), a protein kinase (Aurora-A), and a ganglioside (GM1). Protocols in this article describe a general procedure for the library construction and the library screening.

Binding affinity of five PBPs to Ostrinia sex pheromones

USDA-ARS?s Scientific Manuscript database

Pheromone binding proteins (PBPs) of Lepidoptera function in chemical communication, mate attraction and recognition, and may be involved in reinforcement of sexual isolation between recently diverged species. Directional selection was previously predicted between PBP3 orthologs of the corn borer si...

Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2.

PubMed Central

Nan, X; Meehan, R R; Bird, A

1993-01-01

MeCP2 is a chromosomal protein which binds to DNA that is methylated at CpG. In situ immunofluorescence in mouse cells has shown that the protein is most concentrated in pericentromeric heterochromatin, suggesting that MeCP2 may play a role in the formation of inert chromatin. Here we have isolated a minimal methyl-CpG binding domain (MBD) from MeCP2. MBD is 85 amino acids in length, and binds exclusively to DNA that contains one or more symmetrically methylated CpGs. MBD has negligable non-specific affinity for DNA, confirming that non-specific and methyl-CpG specific binding domains of MeCP2 are distinct. In vitro footprinting indicates that MBD binding can protect a 12 nucleotide region surrounding a methyl-CpG pair, with an approximate dissociation constant of 10(-9) M. Images PMID:8177735

Comparative studies of human and chicken retinol-binding proteins and prealbumins.

PubMed

Kopelman, M; Mokady, S; Cogan, U

1976-08-09

Microheterogeneity of retinol-binding proteins of human plasma and urine, and of chicken plasma was studied by polyacrylamide gel electrophoresis. All three protein systems were found microheterogenous. Incorporation of retinol into the protein preparations on the one hand, and depletion of these proteins from retinol on the other hand, enabled us to clarify the extent to which the presence or absence of the ligand affects the apparent heterogeneity. Upon electrophoresis, each of the native proteins displayed two pairs of protein zones. It appeared that within each pair the fast moving band corresponded to aporetinol-binding protein which upon binding of retinol was converted to a holoprotein with a slightly lower mobility. However, it did not seem that proteins of one pair were converted to proteins of the second pair upon binding of retinol, substantiating ghe microheterogenous character of this protein system. A rapid, two step procedure for isolation of prealbumins from plasma is described. The method which consists of DEAE-cellulose chromatography follwed by preparative electrophoresis was utilized to separate human and chicken prealbumins. Routine dodecyl sulphate electrophoresis resulted in partial dissociation of human prealbumin but in no dissociation of the chicken protein. More drastic treatments prior to electrophoresis were needed to effect complete disruption of both proteins into subunits.

Interaction of sucralose with whey protein: Experimental and molecular modeling studies

NASA Astrophysics Data System (ADS)

Zhang, Hongmei; Sun, Shixin; Wang, Yanqing; Cao, Jian

2017-12-01

The objective of this research was to study the interactions of sucralose with whey protein isolate (WPI) by using the three-dimensional fluorescence spectroscopy, circular dichroism spectroscopy and molecular modeling. The results showed that the peptide strands structure of WPI had been changed by sucralose. Sucralose binding induced the secondary structural changes and increased content of aperiodic structure of WPI. Sucralose decreased the thermal stability of WPI and acted as a structure destabilizer during the thermal unfolding process of protein. In addition, the existence of sucralose decreased the reversibility of the unfolding of WPI. Nonetheless, sucralose-WPI complex was less stable than protein alone. The molecular modeling result showed that van der Waals and hydrogen bonding interactions contribute to the complexation free binding energy. There are more than one possible binding sites of WPI with sucralose by surface binding mode.

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

PubMed

Salceda, Rocío; Aguirre-Ramirez, Marisela

2005-03-01

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

Molecular cloning and expression analysis of rainbow trout (Oncorhynchus mykiss)CCAAT/enhancer binding protein genes and their responses to induction by GH in vitro and in vivo

USDA-ARS?s Scientific Manuscript database

CCAAT/enhancer-binding proteins (C/EBPs) are transcription factors consisting of six isoforms and play diverse physiological roles in vertebrates. In rainbow trout (Oncorhynchus mykiss), in addition to the reported C/EBPbeta1,we have isolated cDNA of four other isoforms, C/EBPalpha, C/EBPbeta2, C/E...

Isolation and Characterization of a Sex-Specific Lectin in a Marine Red Alga, Aglaothamnion oosumiense Itono

PubMed Central

Han, Jong Won; Klochkova, Tatyana A.; Shim, Jun Bo; Yoon, Kangsup

2012-01-01

In red algae, spermatial binding to female trichogynes is mediated by a lectin-carbohydrate complementary system. Aglaothamnion oosumiense is a microscopic filamentous red alga. The gamete recognition and binding occur at the surface of the hairlike trichogyne on the female carpogonium. Male spermatia are nonmotile. Previous studies suggested the presence of a lectin responsible for gamete recognition on the surface of female trychogynes. A novel N-acetyl-d-galactosamine-specific protein was isolated from female plants of A. oosumiense by affinity chromatography and named AOL1. The lectin was monomeric and did not agglutinate horse blood or human erythrocytes. The N-terminal amino acid sequence of the protein was analyzed, and degenerate primers were designed. A full-length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends-PCR (RACE-PCR). The cDNA was 1,095 bp in length and coded for a protein of 259 amino acids with a deduced molecular mass of 21.4 kDa, which agreed well with the protein data. PCR analysis using genomic DNA showed that both male and female plants have this gene. However, Northern blotting and two-dimensional electrophoresis showed that this protein was expressed 12 to 15 times more in female plants. The lectin inhibited spermatial binding to the trichogynes when preincubated with spermatia, suggesting its involvement in gamete binding. PMID:22865077

EV-Associated MMP9 in High-Grade Serous Ovarian Cancer Is Preferentially Localized to Annexin V-Binding EVs.

PubMed

Reiner, Agnes T; Tan, Sisareuth; Agreiter, Christiane; Auer, Katharina; Bachmayr-Heyda, Anna; Aust, Stefanie; Pecha, Nina; Mandorfer, Mattias; Pils, Dietmar; Brisson, Alain R; Zeillinger, Robert; Lim, Sai Kiang

2017-01-01

High-grade serous ovarian cancer (HGSOC) is the most aggressive type of ovarian cancer and is responsible for most deaths caused by gynecological cancers. Numerous candidate biomarkers were identified for this disease in the last decades, but most were not sensitive or specific enough for clinical applications. Hence, new biomarkers for HGSOC are urgently required. This study aimed to identify new markers by isolating different extracellular vesicle (EV) types from the ascites of ovarian cancer patients according to their affinities for lipid-binding proteins and analyzing their protein cargo. This approach circumvents the low signal-to-noise ratio when using biological fluids for biomarker discovery and the issue of contamination by large non-EV complexes. We isolated and analyzed three distinct EV populations from the ascites of patients with ovarian cancer or cirrhosis and observed that Annexin V-binding EVs have higher levels of matrix metalloproteinase 9 in malignant compared to portal-hypertensive ascites. As this protein was not detected in other EV populations, this study validates our approach of using different EV types for optimal biomarker discovery. Furthermore, MMP9 in Annexin V-binding EVs could be a HGSOC biomarker with enhanced specificity, because its identification requires detection of two distinct components, that is, lipid and protein.

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

NASA Technical Reports Server (NTRS)

Safadi, F.; Reddy, V. S.; Reddy, A. S.

2000-01-01

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

Isolation and partial characterisation of human riboflavin carrier protein and the estimation of its levels during human pregnancy.

PubMed

Natraj, U; George, S; Kadam, P

1988-06-01

Human cord serum contains protein(s) capable of binding to [14C]-riboflavin. Riboflavin-bound protein cross-reacts with anti-serum to chicken riboflavin carrier protein (cRCP). The carrier protein was isolated using affinity chromatography on a riboflavin AH Sepharose column. Bulk isolation and purification was also attempted by a combination of ion exchange, gel filtration and gel permeation chromatography on HPLC. The protein so isolated had a molecular weight of 36,000 +/- 2,000 daltons with an isoelectric point of 4.1. The levels of RCP in maternal serum during pregnancy were monitored using a sensitive heterologous radioimmunoassay system, using cRCP as standard and anti serum to cRCP. The levels of the protein increased after 4 months and remained significantly elevated up to 8 months. Although the level of the protein in the maternal serum remained low until 4 months, its level in amniotic fluid was elevated 2-3-fold as compared to that in serum.

Development of a Dehalogenase-Based Protein Fusion Tag Capable of Rapid, Selective and Covalent Attachment to Customizable Ligands

PubMed Central

Encell, Lance P; Friedman Ohana, Rachel; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Wood, Monika G; Los, Georgyi V; McDougall, Mark G; Zimprich, Chad; Karassina, Natasha; Learish, Randall D; Hurst, Robin; Hartnett, James; Wheeler, Sarah; Stecha, Pete; English, Jami; Zhao, Kate; Mendez, Jacqui; Benink, Hélène A; Murphy, Nancy; Daniels, Danette L; Slater, Michael R; Urh, Marjeta; Darzins, Aldis; Klaubert, Dieter H; Bulleit, Robert F; Wood, Keith V

2012-01-01

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins. PMID:23248739

[Molecular organization of glutamate-sensitive chemoexcitatory membranes of nerve cells. Comparative analysis of glutamate-binding membrane proteins from the cerebral cortex of rats and humans].

PubMed

Dambinova, S A; Gorodinskiĭ, A I; Lekomtseva, T M; Koreshonkov, O N

1987-10-01

The kinetics of 3H-L-glutamate binding to human brain synaptic membranes revealed the existence of one type of binding sites with Kd and Vmax comparable with those for freshly isolated rat brain membranes. The fraction of glutamate-binding proteins (GBP) was shown to contain three components with Mr of 14, 60 and 280 kD whose stoichiometry is specific for human and rat brain. All fractions were found to bind the radiolabeled neurotransmitter and to dissociate into subunits with Mr of 14 kD after treatment with-potent detergents (with the exception of the 56-60 kD component). Study of association-dissociation of GBP protein subunits by high performance liquid chromatography confirmed the hypothesis on the oligomeric structure of glutamate receptors which are made up of low molecular weight glycoprotein-lipid subunits and which form ionic channels by way of repeated association. Despite the similarity of antigen determinants in the active center of glutamate receptors from human and rat brain, it was assumed that the stoichiometry of structural organization of receptor subunits isolated from different sources is different. The functional role of structural complexity of human brain glutamate receptors is discussed.

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.

PubMed

Heitner, Tara; Satozawa, Noboru; McLean, Kirk; Vogel, David; Cobb, Ronald R; Liu, Bing; Mahmoudi, Mithra; Finster, Silke; Larsen, Brent; Zhu, Ying; Zhou, Hongxing; Müller-Tiemann, Beate; Monteclaro, Felipe; Zhao, Xiao-Yan; Light, David R

2006-12-01

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

Stable Binding of Alternative Protein-enriched Food Matrices with Concentrated Cranberry Bioflavonoids for Functional Food Applications

PubMed Central

Grace, Mary H.; Guzman, Ivette; Roopchand, Diana E.; Moskal, Kristin; Cheng, Diana M.; Pogrebnyak, Natasha; Raskin, Ilya; Howell, Amy; Lila, Mary Ann

2013-01-01

Defatted soy flour (DSF), soy protein isolate (SPI), hemp protein isolate (HPI), medium roast peanut flour (MPF) and pea protein isolate (PPI) stably bind and concentrate cranberry (CB) polyphenols, creating protein/polyphenol-enriched matrices. Proanthocyanidins (PAC) in the enriched matrices ranged from 20.75 mg/g (CB-HPI) to 10.68 mg/g (CB-SPI). Anthocyanins (ANC) ranged from 3.19 mg/g (CB-DSF) to 1.68 mg/g (CB-SPI), while total phenolics (TP) ranged from 37.61 mg/g (CB-HPI) to 21.29 mg/g (CB-SPI). LC-MS indicated that the enriched matrices contained all identifiable ANC, PAC and flavonols present in CB juice. Complexation with SPI stabilized and preserved the integrity of the CB polyphenolic components for at least 15 weeks at 37 °C. PAC isolated from enriched matrices demonstrated comparable anti-adhesion bioactivity to PAC isolated directly from CB juice (MIC 0.4 to 0.16 mg/mL), indicating their potential utility for maintenance of urinary tract health. Approximately 1.0 g of polyphenol-enriched matrix delivered the same amount of PAC available in one cup (300 mL) of commercial CB juice cocktail; which has been shown clinically to be the prophylactic dose for reducing recurring urinary tract infections. CB-SPI inhibited gram- positive and gram-negative bacterial growth. Nutritional and sensory analyses indicated that the targeted CB-matrix combinations have high potential for incorporation in functional food formulations. PMID:23786629

Binding, stability, and antioxidant activity of quercetin with soy protein isolate particles.

PubMed

Wang, Yufang; Wang, Xiaoyong

2015-12-01

This work is to study the potential of particles fabricated from soy protein isolate (SPI) as a protective carrier for quercetin. When the concentration of SPI particles increases from 0 to 0.35 g/L, quercetin gives a gradually increased fluorescence intensity and fluorescence anisotropy. The addition of quercetin can highly quench the intrinsic fluorescence of SPI particles. These results are explained in terms of the binding of quercetin to the hydrophobic pockets of SPI particles mainly through the hydrophobic force together with the hydrogen bonding. The small difference in the binding constants at 25 and 40 °C suggests the structural stability of SPI particles. The relative changes in values of Gibbs energy, enthalpy, and entropy indicate that the binding of quercetin with SPI particles is spontaneous and hydrophobic interaction is the major force. Furthermore, SPI particles are superior to native SPI for improving the stability and radical scavenging activity of quercetin. Copyright © 2015 Elsevier Ltd. All rights reserved.

Plasmodium vivax Invasion of Human Erythrocytes Inhibited by Antibodies Directed against the Duffy Binding Protein

PubMed Central

Grimberg, Brian T; Udomsangpetch, Rachanee; Xainli, Jia; McHenry, Amy; Panichakul, Tasanee; Sattabongkot, Jetsumon; Cui, Liwang; Bockarie, Moses; Chitnis, Chetan; Adams, John; Zimmerman, Peter A; King, Christopher L

2007-01-01

Background Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes. Methods and Findings Using a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax–exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax–exposed people reduced P. vivax invasion by up to 54%. Conclusions These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection. PMID:18092885

Albizia lebbeck Seed Coat Proteins Bind to Chitin and Act as a Defense against Cowpea Weevil Callosobruchus maculatus.

PubMed

Silva, Nadia C M; De Sá, Leonardo F R; Oliveira, Eduardo A G; Costa, Monique N; Ferreira, Andre T S; Perales, Jonas; Fernandes, Kátia V S; Xavier-Filho, Jose; Oliveira, Antonia E A

2016-05-11

The seed coat is an external tissue that participates in defense against insects. In some nonhost seeds, including Albizia lebbeck, the insect Callosobruchus maculatus dies during seed coat penetration. We investigated the toxicity of A. lebbeck seed coat proteins to C. maculatus. A chitin-binding protein fraction was isolated from seed coat, and mass spectrometry showed similarity to a C1 cysteine protease. By ELM program an N-glycosylation interaction motif was identified in this protein, and by molecular docking the potential to interact with N-acetylglucosamine (NAG) was shown. The chitin-binding protein fraction was toxic to C. maculatus and was present in larval midgut and feces but not able to hydrolyze larval gut proteins. It did not interfere, though, with the intestinal cell permeability. These results indicate that the toxicity mechanism of this seed coat fraction may be related to its binding to chitin, present in the larvae gut, disturbing nutrient absorption.

Antascomicins A, B, C, D and E. Novel FKBP12 binding compounds from a Micromonospora strain.

PubMed

Fehr, T; Sanglier, J J; Schuler, W; Gschwind, L; Ponelle, M; Schilling, W; Wioland, C

1996-03-01

5 novel ascomycin-like compounds, antascomicins A, B, C, D and E were isolated from a strain of Micromonospora. The antascomicins bind strongly to the FK506-binding protein FKBP12 and antagonize the immunosuppressive activity of FK506 and rapamycin. The strain description, fermentation, structure elucidation and biological activity of these compounds are described.

Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion.

PubMed

Younger, Andrew K D; Su, Peter Y; Shepard, Andrea J; Udani, Shreya V; Cybulski, Thaddeus R; Tyo, Keith E J; Leonard, Joshua N

2018-02-01

Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors. To develop and evaluate the BERDI method, we generated a library of candidate biosensors in which a zinc finger DNA-binding domain was inserted into maltose binding protein, which served as a model well-studied metabolite-binding protein. Library diversity was characterized by several methods, a selection scheme was deployed, and ultimately several distinct and functional maltose-responsive transcriptional biosensors were identified. We hypothesize that the BERDI method comprises a generalizable strategy that may ultimately be applied to convert a wide range of metabolite-binding proteins into novel biosensors for applications in metabolic engineering and synthetic biology. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Pathophysiology and Toxicokinetic Studies of Blue-Green Algae Intoxication in the Swine Model

DTIC Science & Technology

1987-08-31

bovine aer, albamin caused similar reductions !u toxicity, suggesting nonspecific protein binding. Saturation at the N-methyldehydroalanine of MCYST-A...need to conserve toxin and the goal of creating a model for naturally occurring toxicosis caused us to evaluate the oral administration of microcystin...the isolated ileal loop preparation. Pancreatic enzymes and protein-binding were examined as 2 possible causes for the low oral toxicity of MCYST-A

DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

NASA Astrophysics Data System (ADS)

Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

1984-08-01

A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

Characterization of mannose binding lectin from channel catfish Ictalurus punctatus

USDA-ARS?s Scientific Manuscript database

Mannose-binding lectin (MBL) is an important component of innate immunity capable of activating the lectin pathway of the complement system. A MBL gene was isolated from channel catfish (Ictalurus punctatus). The deduced protein contains a canonical collagen-like domain, a carbohydrate recognition d...

Imipenem Resistance in Clostridium difficile Ribotype 017, Portugal

PubMed Central

Isidro, Joana; Santos, Andrea; Nunes, Alexandra; Borges, Vítor; Silva, Catarina; Vieira, Luís; Mendes, Aristides L.; Serrano, Mónica; Henriques, Adriano O.; Gomes, João Paulo

2018-01-01

We describe imipenem-resistant and imipenem-susceptible clinical isolates of Clostridium difficile ribotype 017 in Portugal. All ribotype 017 isolates carried an extra penicillin-binding protein gene, pbp5, and the imipenem-resistant isolates had additional substitutions near the transpeptidase active sites of pbp1 and pbp3. These clones could disseminate and contribute to imipenem resistance. PMID:29553322

Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

NASA Technical Reports Server (NTRS)

Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

1995-01-01

The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

Synthesis of novel (-)-epicatechin derivatives as potential endothelial GPER agonists: Evaluation of biological effects.

PubMed

Sarmiento, Viviana; Ramirez-Sanchez, Israel; Moreno-Ulloa, Aldo; Romero-Perez, Diego; Chávez, Daniel; Ortiz, Miguel; Najera, Nayelli; Correa-Basurto, Jose; Villarreal, Francisco; Ceballos, Guillermo

2018-02-15

To potentially identify proteins that interact (i.e. bind) and may contribute to mediate (-)-epicatechin (Epi) responses in endothelial cells we implemented the following strategy: 1) synthesis of novel Epi derivatives amenable to affinity column use, 2) in silico molecular docking studies of the novel derivatives on G protein-coupled estrogen receptor (GPER), 3) biological assessment of the derivatives on NO production, 4) implementation of an immobilized Epi derivative affinity column and, 5) affinity column based isolation of Epi interacting proteins from endothelial cell protein extracts. For these purposes, the Epi phenol and C3 hydroxyl groups were chemically modified with propargyl or mesyl groups. Docking studies of the novel Epi derivatives on GPER conformers at 14 ns and 70 ns demostrated favorable thermodynamic interactions reaching the binding site. Cultures of bovine coronary artery endothelial cells (BCAEC) treated with Epi derivatives stimulated NO production via Ser1179 phosphorylation of eNOS, effects that were attenuated by the use of the GPER blocker, G15. Epi derivative affinity columns yielded multiple proteins from BCAEC. Proteins were electrophoretically separated and inmmunoblotting analysis revealed GPER as an Epi derivative binding protein. Altogether, these results validate the proposed strategy to potentially isolate and identify novel Epi receptors that may account for its biological activity. Copyright © 2018 Elsevier Ltd. All rights reserved.

The 87-kD A gamma-globin enhancer-binding protein is a product of the HOXB2(HOX2H) locus.

PubMed

Sengupta, P K; Lavelle, D E; DeSimone, J

1994-03-01

Developmental regulation of globin gene expression may be controlled by developmental stage-specific nuclear proteins that influence interactions between the locus control region and local regulatory sequences near individual globin genes. We previously isolated an 87-kD nuclear protein from K562 cells that bound to DNA sequences in the beta-globin locus control region, gamma-globin promoter, and A gamma-globin enhancer. The presence of this protein in fetal globin-expressing cells and its absence in adult globin-expressing cells suggested that it may be a developmental stage-specific factor. A lambda gt11 K562 cDNA clone encoding a portion of the HOXB2 (formerly HOX2H) homeobox gene was isolated on the basis of the ability of its beta-galactosidase fusion protein to bind to the same DNA sequences as the 87-kD K562 protein. Because no other relationship had been established between the 87-kD K562 protein and the HOXB2 protein other than their ability to bind ot the same DNA sequences, we have investigated whether the two proteins are related antigenically. Our data show that antisera produced against the HOXB2-beta-gal fusion protein and a synthetic HOXB2 decapeptide react specifically with an 87-kD protein from K562 nuclear extract, showing that the 87-kD K562 nuclear protein is a product of the HOXB2 locus, and is the first demonstration of cellular HOXB2 protein.

Identification of RNAIII-binding proteins in Staphylococcus aureus using tethered RNAs and streptavidin aptamers based pull-down assay.

PubMed

Zhang, Xu; Zhu, Qing; Tian, Tian; Zhao, Changlong; Zang, Jianye; Xue, Ting; Sun, Baolin

2015-05-15

It has been widely recognized that small RNAs (sRNAs) play important roles in physiology and virulence control in bacteria. In Staphylococcus aureus, many sRNAs have been identified and some of them have been functionally studied. Since it is difficult to identify RNA-binding proteins (RBPs), very little has been known about the RBPs in S. aureus, especially those associated with sRNAs. Here we adopted a tRNA scaffold streptavidin aptamer based pull-down assay to identify RBPs in S. aureus. The tethered RNA was successfully captured by the streptavidin magnetic beads, and proteins binding to RNAIII were isolated and analyzed by mass spectrometry. We have identified 81 proteins, and expressed heterologously 9 of them in Escherichia coli. The binding ability of the recombinant proteins with RNAIII was further analyzed by electrophoresis mobility shift assay, and the result indicates that proteins CshA, RNase J2, Era, Hu, WalR, Pyk, and FtsZ can bind to RNAIII. This study suggests that some proteins can bind to RNA III in S. aureus, and may be involved in RNA III function. And tRSA based pull-down assay is an effective method to search for RBPs in bacteria, which should facilitate the identification and functional study of RBPs in diverse bacterial species.

The amino-terminal region of the retinoblastoma gene product binds a novel nuclear matrix protein that co-localizes to centers for RNA processing

PubMed Central

1994-01-01

The tumor suppressing capacity of the retinoblastoma protein (p110RB) is dependent on interactions made with cellular proteins through its carboxy-terminal domains. How the p110RB amino-terminal region contributes to this activity is unclear, though evidence now indicates it is important for both growth suppression and regulation of the full- length protein. We have used the yeast two-hybrid system to screen for cellular proteins which bind to the first 300 amino acids of p110RB. The only gene isolated from this screen encodes a novel 84-kD nuclear matrix protein that localizes to subnuclear regions associated with RNA processing. This protein, p84, requires a structurally defined domain in the amino terminus of p110RB for binding. Furthermore, both in vivo and in vitro experiments demonstrate that p84 binds preferentially to the functionally active, hypophosphorylated form of p110RB. Thus, the amino terminus of p110RB may function in part to facilitate the binding of growth promoting factors at subnuclear regions actively involved in RNA metabolism. PMID:7525595

Discovery and application of peptides that bind to proteins and solid state inorganic materials

NASA Astrophysics Data System (ADS)

Stearns, Linda A.

A series of three projects was undertaken on the theme of peptide-based molecular recognition. In the first project, a messenger RNA (mRNA) display selection was carried out against the II-VI semiconductors zinc sulfide (ZnS), zinc selenide (ZnSe), and cadmium sulfide (CdS). Sequence analysis of 18-mer semiconductor-binding peptides (SBPs) following four rounds of selection indicated that the amino acid sequences were enriched in polar residues compared to the naive library, suggesting that hydrogen-bonding interactions are a dominant mode of interaction between the SBPs and their cognate inorganic surfaces. Select peptides were expressed as fusions of the green fluorescent protein (GFP) to visualize their recognition of semiconductor crystals. Interpretation of the results was complicated by a high fluorescence background that was observed with certain control GFP fusions. Additional experiments, including cross-specificity binding assays, are needed to characterize the peptides that were isolated in this selection. A second project described the practical application of a known inorganic-binding and nucleating peptide. Peptide A3, which was previously isolated by phage display, was chemically conjugated to a short DNA strand using the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). The resulting peptide-DNA conjugate was hybridized to ten complementary single-stranded capture probes extending outward from the surface of an origami DNA nanotube. A gold precursor solution was added to initiate nucleation and growth of gold nanoparticles at the site of the peptide. Transmission electron microscopy (TEM) was used to visualize the gold nanoparticle-decorated nanostructures. This approach holds immense promise for organizing compositionally-diverse materials at the nanoscale. In a third project, a novel non-iterative approach to mRNA display called covalent capture was demonstrated. Using human transferrin as a target protein, peptides with low-nanomolar affinity were isolated from a combinatorial library of one trillion distinct 12-mer peptide sequences by using UV light to covalently crosslink the peptides to a photoreactive arm that was displayed on the protein surface. The best peptide isolated from this screen exhibited a binding affinity constant (Kd) of 3 nM, which is equivalent to some of the best peptides isolated after many rounds of traditional bead-based selection. The approach itself is general and could be applied to many different types of problems in molecular biology.

Isolation and functional characterization of CE1 binding proteins.

PubMed

Lee, Sun-ji; Park, Ji Hye; Lee, Mi Hun; Yu, Ji-hyun; Kim, Soo Young

2010-12-16

Abscisic acid (ABA) is a plant hormone that controls seed germination, protective responses to various abiotic stresses and seed maturation. The ABA-dependent processes entail changes in gene expression. Numerous genes are regulated by ABA, and promoter analyses of the genes revealed that cis-elements sharing the ACGTGGC consensus sequence are ubiquitous among ABA-regulated gene promoters. The importance of the core sequence, which is generally known as ABA response element (ABRE), has been demonstrated by various experiments, and its cognate transcription factors known as ABFs/AREBs have been identified. Although necessary, ABRE alone is not sufficient, and another cis-element known as "coupling element (CE)" is required for full range ABA-regulation of gene expression. Several CEs are known. However, despite their importance, the cognate transcription factors mediating ABA response via CEs have not been reported to date. Here, we report the isolation of transcription factors that bind one of the coupling elements, CE1. To isolate CE1 binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing a trimer of the CE1 element were prepared and introduced into a yeast strain. The yeast was transformed with library DNA that represents RNA isolated from ABA-treated Arabidopsis seedlings. From the screen of 3.6 million yeast transformants, we isolated 78 positive clones. Analysis of the clones revealed that a group of AP2/ERF domain proteins binds the CE1 element. We investigated their expression patterns and analyzed their overexpression lines to investigate the in vivo functions of the CE element binding factors (CEBFs). Here, we show that one of the CEBFs, AtERF13, confers ABA hypersensitivity in Arabidopsis, whereas two other CEBFs enhance sugar sensitivity. Our results indicate that a group of AP2/ERF superfamily proteins interacts with CE1. Several CEBFs are known to mediate defense or abiotic stress response, but the physiological functions of other CEBFs remain to be determined. Our in vivo functional analysis of several CEBFs suggests that they are likely to be involved in ABA and/or sugar response. Together with previous results reported by others, our current data raise an interesting possibility that the coupling element CE1 may function not only as an ABRE but also as an element mediating biotic and abiotic stress responses.

Plasma membrane isolation using immobilized concanavalin A magnetic beads.

PubMed

Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

2012-01-01

Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

Quinupristin-Dalfopristin Resistance in Streptococcus pneumoniae: Novel L22 Ribosomal Protein Mutation in Two Clinical Isolates from the SENTRY Antimicrobial Surveillance Program

PubMed Central

Jones, Ronald N.; Farrell, David J.; Morrissey, Ian

2003-01-01

Resistance to quinupristin-dalfopristin (Q/D) among gram-positive cocci has been very uncommon. Two clinical isolates among 8,837 (0.02%) Streptococcus pneumoniae isolates were discovered in 2001 to 2002 with Q/D MICs of 4 μg/ml. Each had a 5-amino-acid tandem duplication (RTAHI) in the L22 ribosomal protein gene (rplV) preventing synergistic ribosomal binding of the streptogramin combination. Similar gene duplication has been reported in Q/D-resistant Staphylococcus aureus. PMID:12878545

Isolation of anticancer drug TAXOL from Pestalotiopsis breviseta with apoptosis and B-Cell lymphoma protein docking studies.

PubMed

Kathiravan, G; Sureban, Sripathi M; Sree, Harsha N; Bhuvaneshwari, V; Kramony, Evelin

2012-12-01

Extraction and investigation of TAXOL from Pestalotiopsis breviseta (Sacc.) using protein docking, which is a computational technique that samples conformations of small molecules in protein-binding sites. Scoring functions are used to assess which of these conformations best complements the protein binding site and active site prediction. Coelomycetous fungi P. breviseta (Sacc.) Steyaert was screened for the production of TAXOL, an anticancer drug. TAXOL PRODUCTION WAS CONFIRMED BY THE FOLLOWING METHODS: Ultraviolet (UV) spectroscopic analysis, Infrared analysis, High performance liquid chromatography analysis (HPLC), and Liquid chromatography mass spectrum (LC-MASS). TAXOL produced by the fungi was compared with authentic TAXOL, and protein docking studies were performed. The BCL2 protein of human origin showed a higher affinity toward the compound paclitaxel. It has the binding energy value of -13.0061 (KJ/Mol) with four hydrogen bonds.

Isolation of anticancer drug TAXOL from Pestalotiopsis breviseta with apoptosis and B-Cell lymphoma protein docking studies

PubMed Central

Kathiravan, G.; Sureban, Sripathi M.; Sree, Harsha N.; Bhuvaneshwari, V.; Kramony, Evelin

2012-01-01

Background: Extraction and investigation of TAXOL from Pestalotiopsis breviseta (Sacc.) using protein docking, which is a computational technique that samples conformations of small molecules in protein-binding sites. Scoring functions are used to assess which of these conformations best complements the protein binding site and active site prediction. Materials and Methods: Coelomycetous fungi P. breviseta (Sacc.) Steyaert was screened for the production of TAXOL, an anticancer drug. Results: TAXOL production was confirmed by the following methods: Ultraviolet (UV) spectroscopic analysis, Infrared analysis, High performance liquid chromatography analysis (HPLC), and Liquid chromatography mass spectrum (LC-MASS). TAXOL produced by the fungi was compared with authentic TAXOL, and protein docking studies were performed. Conclusion: The BCL2 protein of human origin showed a higher affinity toward the compound paclitaxel. It has the binding energy value of −13.0061 (KJ/Mol) with four hydrogen bonds. PMID:24808664

[Experiences with an isolation method for retinal S-antigen and interphotoreceptor retinoid-binding protein].

PubMed

Sych, F J; Strobel, J

1996-12-01

S-antigen (AgS) and interphotoreceptor retinoid-binding protein (IRBP) are two highly potent uveitopathogenic autoantigens of the retina frequently used to trigger autoimmune uveitis in animal trials. The aim of this study was the simultaneous isolation of both proteins from bovine retina, additionally avoiding time-consuming dialysis and employing prepacked, commercially available cartridges. Retina extract, obtained in the usual manner, was precipitated with ammonium sulfate. Both the desalting and the buffer exchange of dissolved precipitate were carried out using a Bio-Gel P6 column. Subsequent separation with Econo-Pac Q gave prepurified AgS and IRBP fractions. Further purification was realized predominantly with Econo-Pac Q, -HTP and -HIC (5-ml cartridges). AgS and IRBP were isolated in high purity (sodium dodecylsulfate polyacrylamide gel electrophoresis, silver stain) from bovine retina using prepacked cartridges with yields of approximately 0.25 (for AgS) and 0.15 (for IRBP) mg/g retina, respectively. The application of ready-to-use cartridges allows simultaneous isolation of AgS and IRBP in milligram amounts under simplified conditions. This approach might be of particular interest for small samples of retina.

Fatty acid uptake by isolated rat heart myocytes represents a carrier-mediated transport process.

PubMed Central

Stremmel, W

1988-01-01

The mechanism by which fatty acids enter cardiomyocytes is unclear. Therefore, the influx kinetics of [3H]oleate into isolated rat heart myocytes were examined. Cells were incubated at 37 degrees C with [3H]oleate bound to albumin in various molar ratios and the initial rate of uptake (V0) was determined as a function of the unbound oleate concentration in the medium. V0 was saturable with increasing oleate concentrations incubated (Km 78 nM; Vmax 1.9 nmol X min-1 per 10(6) cells) and temperature dependent with an optimum at 37 degrees C. Furthermore, binding of [3H]oleate to isolated plasma membranes of cardiomyocytes was saturable, revealing a KD of 42 nM, and was inhibited by heat denaturation or trypsin pretreatment of the membranes. From these membranes a single 40-kD protein with high affinity for a variety of long chain fatty acids was isolated. With a monospecific antibody to this membrane protein, binding as well as cellular influx of [3H]oleate was selectively inhibited. These data indicate that at least a portion of myocardial fatty acid uptake is mediated by a specific membrane protein. Images PMID:3343344

GFP tagging of sieve element occlusion (SEO) proteins results in green fluorescent forisomes.

PubMed

Pélissier, Hélène C; Peters, Winfried S; Collier, Ray; van Bel, Aart J E; Knoblauch, Michael

2008-11-01

Forisomes are Ca(2+)-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as 'FOR' proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca(2+) and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that 'FOR'-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca(2+) binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.

GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

PubMed Central

Pélissier, Hélène C.; Peters, Winfried S.; Collier, Ray; van Bel, Aart J. E.; Knoblauch, Michael

2008-01-01

Forisomes are Ca2+-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as ‘FOR’ proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca2+ and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that ‘FOR’-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca2+ binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism. PMID:18784195

Identification of a collagen type I adhesin of Bacteroides fragilis.

PubMed

Galvão, Bruna P G V; Weber, Brandon W; Rafudeen, Mohamed S; Ferreira, Eliane O; Patrick, Sheila; Abratt, Valerie R

2014-01-01

Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼ 31 and ∼ 34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼ 31 kDa and the ∼ 34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼ 31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein.

Identification of a Collagen Type I Adhesin of Bacteroides fragilis

PubMed Central

Galvão, Bruna P. G. V.; Weber, Brandon W.; Rafudeen, Mohamed S.; Ferreira, Eliane O.; Patrick, Sheila; Abratt, Valerie R.

2014-01-01

Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼31 and ∼34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼31 kDa and the ∼34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein. PMID:24618940

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

PubMed

Lämmler, C

1988-08-01

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

Investigation of the inhibitors of histone-lysine N-methyltransferase SETD2 for acute lymphoblastic leukaemia from traditional Chinese medicine.

PubMed

Chang, Y-L; Chen, H-Y; Chen, K-B; Chen, K-C; Chang, K-L; Chang, P-C; Chang, T-T; Chen, Y-C

2016-07-01

Leukaemia is the leading cause of childhood malignancies. Recent research indicates that the SETD2 gene is associated with acute lymphoblastic leukaemia. This study aims to identify potential lead compounds from traditional Chinese medicine (TCM) using virtual screening for SET domain containing 2 (SETD2) protein against acute lymphoblastic leukaemia. Docking simulation was performed to determine potential candidates which obtain suitable docking poses in the binding domain of the SETD2 protein. We also performed molecular dynamics (MD) simulation to investigate the stability of docking poses of SETD2 protein complexes with the top three TCM candidates and a control. According to the results of docking and MD simulation, coniselin and coniferyl ferulate have high binding affinity and stable interactions with the SETD2 protein. Coniselin is isolated from the alcoholic extract of Comiselinum vaginatum Thell. Coniferyl ferulate can be isolated from Angelica sinensis, Poria cocos (Schw.) Wolf, and Notopterygium forbesii. Although S-adenosyl-L-homocysteine has more stable interactions with key residues in the binding domain than coniselin and coniferyl ferulate during MD simulation, the TCM compounds coniselin and coniferyl ferulate are still potential candidates as lead compounds for further study in the drug development process with the SETD2 protein against acute lymphoblastic leukaemia.

Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis).

PubMed

Darville, Lancia N F; Merchant, Mark E; Maccha, Venkata; Siddavarapu, Vivekananda Reddy; Hasan, Azeem; Murray, Kermit K

2012-02-01

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35kDa protein was ~98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates. Copyright © 2011 Elsevier Inc. All rights reserved.

Gene Isolation Using Degenerate Primers Targeting Protein Motif: A Laboratory Exercise

ERIC Educational Resources Information Center

Yeo, Brandon Pei Hui; Foong, Lian Chee; Tam, Sheh May; Lee, Vivian; Hwang, Siaw San

2018-01-01

Structures and functions of protein motifs are widely included in many biology-based course syllabi. However, little emphasis is placed to link this knowledge to applications in biotechnology to enhance the learning experience. Here, the conserved motifs of nucleotide binding site-leucine rich repeats (NBS-LRR) proteins, successfully used for the…

Delineation of Methyl-DNA Binding Protein Interactions in the Prostate Cancer Genome

DTIC Science & Technology

2013-07-01

smaller portion was used for isolation of mRNAs - see below.) Chromatin immunoprecipitations were carried out using antibodies that recognize MeCP2...tissues were performed with antibodies directed against MeCP2, MBD1, MBD2 and MBD4 (methyl-CpG binding domain containing proteins) - “ChIP DNA” from...the matched tissues and these antibodies . - The analysis of NGS data was initiated. Differences in the MBD association profiles between matched

Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate.

PubMed

Horne, Irene; Sutherland, Tara D; Harcourt, Rebecca L; Russell, Robyn J; Oakeshott, John G

2002-07-01

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k(cat) than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.

Avian pathogenic Escherichia coli bind fibronectin and laminin.

PubMed

Ramírez, Rosa María; Almanza, Yolanda; González, Rafael; García, Santos; Heredia, Norma

2009-04-01

Avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. We investigated the ability of an isolate of avian pathogenic Escherichia coli to bind fibronectin and laminin. Using Far-western dot blot analysis, we demonstrated the ability of this microorganism to bind basement membrane proteins fibronectin and laminin. Results from an ELISA-based approach indicate that the binding to these membrane proteins was bacterial-dose dependent. Furthermore, two specific E. coli polypeptides, of 32 kDa and 130 kDa, reacted with laminin and fibronectin, respectively. Further evaluation of these potential bacterial adhesins may provide insights into the pathogenesis of colibacillosis.

A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

NASA Technical Reports Server (NTRS)

Yang, Tianbao; Poovaiah, B. W.

2002-01-01

We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

PubMed Central

Galloway-Peña, Jessica R.; Liang, Xiaowen; Singh, Kavindra V.; Yadav, Puja; Chang, Chungyu; La Rosa, Sabina Leanti; Shelburne, Samuel; Ton-That, Hung; Höök, Magnus

2014-01-01

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species. PMID:25512313

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

PubMed

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

1992-09-08

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

The human escort protein Hep binds to the ATPase domain of mitochondrial hsp70 and regulates ATP hydrolysis.

PubMed

Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J

2008-09-19

Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8+/-0.2 x 10(-4) s(-1)) at 25 degrees C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain.

The Human Escort Protein Hep Binds to the ATPase Domain of Mitochondrial Hsp70 and Regulates ATP Hydrolysis*

PubMed Central

Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J.

2008-01-01

Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8 ± 0.2 × 10-4 s-1) at 25 °C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain. PMID:18632665

Sequence variations and protein expression levels of the two immune evasion proteins Gpm1 and Pra1 influence virulence of clinical Candida albicans isolates.

PubMed

Luo, Shanshan; Hipler, Uta-Christina; Münzberg, Christin; Skerka, Christine; Zipfel, Peter F

2015-01-01

Candida albicans, the important human fungal pathogen uses multiple evasion strategies to control, modulate and inhibit host complement and innate immune attack. Clinical C. albicans strains vary in pathogenicity and in serum resistance, in this work we analyzed sequence polymorphisms and variations in the expression levels of two central fungal complement evasion proteins, Gpm1 (phosphoglycerate mutase 1) and Pra1 (pH-regulated antigen 1) in thirteen clinical C. albicans isolates. Four nucleotide (nt) exchanges, all representing synonymous exchanges, were identified within the 747-nt long GPM1 gene. For the 900-nt long PRA1 gene, sixteen nucleotide exchanges were identified, which represented synonymous, as well as non-synonymous exchanges. All thirteen clinical isolates had a homozygous exchange (A to G) at position 73 of the PRA1 gene. Surface levels of Gpm1 varied by 8.2, and Pra1 levels by 3.3 fold in thirteen tested isolates and these differences influenced fungal immune fitness. The high Gpm1/Pra1 expressing candida strains bound the three human immune regulators more efficiently, than the low expression strains. The difference was 44% for Factor H binding, 51% for C4BP binding and 23% for plasminogen binding. This higher Gpm1/Pra1 expressing strains result in enhanced survival upon challenge with complement active, Factor H depleted human serum (difference 40%). In addition adhesion to and infection of human endothelial cells was increased (difference 60%), and C3b surface deposition was less effective (difference 27%). Thus, variable expression levels of central immune evasion protein influences immune fitness of the human fungal pathogen C. albicans and thus contribute to fungal virulence.

Resolution of the diadenosine 5',5"'-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase alpha.

PubMed Central

Baril, E; Bonin, P; Burstein, D; Mara, K; Zamecnik, P

1983-01-01

A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A. Images PMID:6576366

Review: Milk Proteins as Nanocarrier Systems for Hydrophobic Nutraceuticals.

PubMed

Kimpel, Florian; Schmitt, Joachim J

2015-11-01

Milk proteins and milk protein aggregates are among the most important nanovehicles in food technology. Milk proteins have various functional properties that facilitate their ability to carry hydrophobic nutraceutical substances. The main functional transport properties that were examined in the reviewed studies are binding of molecules or ions, surface activity, aggregation, gelation, and interaction with other polymers. Hydrophobic binding has been investigated using caseins and isolated β-casein as well as whey proteins. Surface activity of caseins has been used to create emulsion-based carrier systems. Furthermore, caseins are able to self-assemble into micelles, which can incorporate molecules. Gelation and interaction with other polymers can be used to encapsulate molecules into protein networks. The release of transported substances mainly depends on pH and swelling behavior of the proteins. The targeted use of nanocarrier systems requires specific knowledge about the binding mechanisms between the proteins and the carried substances in a certain food matrix. © 2015 Institute of Food Technologists®

Selective Sensitization of Zinc Finger Protein Oxidation by Reactive Oxygen Species through Arsenic Binding*

PubMed Central

Zhou, Xixi; Cooper, Karen L.; Sun, Xi; Liu, Ke J.; Hudson, Laurie G.

2015-01-01

Cysteine oxidation induced by reactive oxygen species (ROS) on redox-sensitive targets such as zinc finger proteins plays a critical role in redox signaling and subsequent biological outcomes. We found that arsenic exposure led to oxidation of certain zinc finger proteins based on arsenic interaction with zinc finger motifs. Analysis of zinc finger proteins isolated from arsenic-exposed cells and zinc finger peptides by mass spectrometry demonstrated preferential oxidation of C3H1 and C4 zinc finger configurations. C2H2 zinc finger proteins that do not bind arsenic were not oxidized by arsenic-generated ROS in the cellular environment. The findings suggest that selectivity in arsenic binding to zinc fingers with three or more cysteines defines the target proteins for oxidation by ROS. This represents a novel mechanism of selective protein oxidation and demonstrates how an environmental factor may sensitize certain target proteins for oxidation, thus altering the oxidation profile and redox regulation. PMID:26063799

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

PubMed

Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

2003-03-01

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.

PubMed

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S; Prasad, Manoj

2011-10-01

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein

PubMed Central

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S

2011-01-01

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncation of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T] [T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein. PMID:21918373

Specific DNA binding of the two chicken Deformed family homeodomain proteins, Chox-1.4 and Chox-a.

PubMed Central

Sasaki, H; Yokoyama, E; Kuroiwa, A

1990-01-01

The cDNA clones encoding two chicken Deformed (Dfd) family homeobox containing genes Chox-1.4 and Chox-a were isolated. Comparison of their amino acid sequences with another chicken Dfd family homeodomain protein and with those of mouse homologues revealed that strong homologies are located in the amino terminal regions and around the homeodomains. Although homologies in other regions were relatively low, some short conserved sequences were also identified. E. coli-made full length proteins were purified and used for the production of specific antibodies and for DNA binding studies. The binding profiles of these proteins to the 5'-leader and 5'-upstream sequences of Chox-1.4 and Chox-a coding regions were analyzed by immunoprecipitation and DNase I footprint assays. These two Chox proteins bound to the same sites in the 5'-flanking sequences of their coding regions with various affinities and their binding affinities to each site were nearly the same. The consensus sequences of the high and low affinity binding sites were TAATGA(C/G) and CTAATTTT, respectively. A clustered binding site was identified in the 5'-upstream of the Chox-a gene, suggesting that this clustered binding site works as a cis-regulatory element for auto- and/or cross-regulation of Chox-a gene expression. Images PMID:1970866

The pig CYP2E1 promoter is activated by COUP-TF1 and HNF-1 and is inhibited by androstenone.

PubMed

Tambyrajah, Winston S; Doran, Elena; Wood, Jeffrey D; McGivan, John D

2004-11-15

Functional analysis of the pig cytochrome P4502E1 (CYP2E1) promoter identified two major activating elements. One corresponded to the hepatic nuclear factor 1 (HNF-1) consensus binding sequence at nucleotides -128/-98 and the other was located in the region -292/-266. The binding of proteins in pig liver nuclear extracts to a synthetic double-stranded oligonucleotide corresponding to this more distal activating sequence was studied by electrophoretic mobility shift assay. The minimum protein binding sequence was identified as TGTTCTGACCTCTGGG. Gel super-shift assays identified the protein binding to this site as chick ovalbumin upstream promoter transcription factor 1 (COUP-TF1). Androstenone inhibited promoter activity in transfection experiments only with constructs which included the COUP-TF1 binding site. Androstenone inhibited COUP-TF1 binding to synthetic oligonucleotides but did not affect HNF-1 binding. The results offer an explanation for the inhibition of CYP2E1 protein expression by androstenone in isolated pig hepatocytes and may be relevant to the low expression of hepatic CYP2E1 in those pigs which accumulate high levels of androstenone in vivo.

Protein FOG--a streptococcal inhibitor of neutrophil function.

PubMed

Johansson, Helena M; Mörgelin, Matthias; Frick, Inga-Maria

2004-12-01

Several strains of group G streptococci (GGS) form aggregates when grown in vitro. Aggregating strains interact with fibrinogen, and this study reports the isolation of a novel self-associating and fibrinogen-binding protein of GGS, denoted protein FOG. Sequencing of the fog gene revealed structural similarity with M proteins of both GGS and group A streptococci (GAS). Analogous to GAS, GGS were found to multiply in human blood. All strains of GGS express protein G, a protein known to interact with the constant region of immunoglobulin G and albumin. Surprisingly, a clinical isolate expressing protein G, but lacking protein FOG, was killed in human whole blood; however, the addition of intact soluble protein FOG restored the ability of the bacteria to survive and multiply in human blood. This is believed to be the first report of a soluble M-like protein salvaging an M-negative strain from being killed. The antibactericidal property of protein FOG is dependent on its fibrinogen-binding activity. Thus, in plasma, FOG precipitates fibrinogen, and when added to whole blood, protein FOG triggers the formation of visible aggregates comprising fibrinogen and neutrophils that are disabled in their killing of the bacteria. Moreover, the results emphasize the importance of an intact FOG molecule, as presented on the bacterial surface, for full protective effect.

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

Nageswara Rao, B.D.; Kemple, M.D.; Prendergast, F.G.

Aequorin is a protein of low molecular weight (20,000) isolated from the jellyfish Aequorea forskalea which emits blue light upon the binding of Ca/sup 2 +/ ions. This bioluminescence requires neither exogenous oxygen nor any other cofactors. The light emission occurs from an excited state of a chromophore (an imidazolopyrazinone) which is tightly and noncovalently bound to the protein. Apparently the binding of Ca/sup 2 +/ by the protein induces changes in the protein conformation which allow oxygen, already bound or otherwise held by the protein, to react with and therein oxidize the chromophore. The resulting discharged protein remains intact,more » with the Ca/sup 2 +/ and the chromophore still bound, but is incapable of further luminescence. The fluorescence spectrum of this discharged protein and the bioluminescence spectrum of the original charged aequorin are identical. A green fluorescent protein (GFP) of approx. 30,000 mol wt isolated from the same organism, functions in vivo as an acceptor of energy from aequorin and subsequently emits green light. We are applying proton nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy to examine structural details of, and fluctuations associated with the luminescent reaction of aequorin and the in vivo energy transfer from aequorin to the GFP.« less

Effect of malondialdehyde modification on the binding of aroma compounds to soy protein isolates.

PubMed

Wang, Juan; Zhao, Mouming; Qiu, Chaoying; Sun, Weizheng

2018-03-01

The interactions of soy protein isolate (SPI) and flavor compounds (hexanal, trans-2-hexenal, 1-octen-3-ol, trans-2-octenal, nonanal, and trans-2-nonenal) were investigated. The influence of SPI structure modified by malondialdehyde (MDA) and flavor compound structure on the interactions were determined by using headspace solid-phase microextraction (SPME) and gas chromatography (GC) combined with mass spectrometry (MS). The binding of native SPI to the flavor compounds decreased in the order trans-2-nonenal>nonanal>trans-2-octenal>trans-2-hexenal>hexanal>1-octen-3-ol. It might be attributed to that aldehydes are more hydrophobic than alcohols. The former is more conducive to hydrophobic binding with the SPI. Furthermore, the aldehydes, in particular trans-s-undecenal, could also react covalently. The effect of MDA modification on protein-flavor interactions depended on the structure of the flavor compound. Upon low concentration of MDA (≤1mM), the binding of all six flavors to SPI increased. However, a further increase in the extent of MDA (≥2.5mM), more soluble and even insoluble aggregates formed, which reduced the binding of hexanal and nonanal to SPI. The other four flavors with double bond revealed little changes in binding (trans-2-octenal, and trans-2-nonenal) or even an increase in binding (trans-2-hexenal, and 1-octen-3-ol). The results suggested that hydrophobic interactions were weakened upon high extent of oxidation, whereas covalent interactions were enhanced. Copyright © 2017 Elsevier Ltd. All rights reserved.

MACROMOLECULAR ADDUCTION BY TRICHLOROACETONITRILE IN THE FISCHER 344 RAT FOLLOWING ORAL GAVAGE

EPA Science Inventory

Male Fischer 344 rats were administered 1- or 2-[14C]-trichloroacetonitrile (TCAN) by ral gavage. NA was isolated from the liver, kidneys and stomach and several protein fractions (globin, albumin and blobulins) were isolated from blood. CAN binds to both the DNA and the blood pr...

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain: Periplasmic Ligand Binding Protein Dret_0059

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

Wu, R.; Wilton, R.; Cuff, M. E.

We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from the Salt Lake Retba in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes butmore » have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.« less

Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.

PubMed Central

Blank, T E; Woods, M P; Lebo, C M; Xin, P; Hopper, J E

1997-01-01

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods. PMID:9111326

Long-lasting, distinct changes in central opioid receptor and urinary bladder functions in models of schizophrenia in rats.

PubMed

Kekesi, Orsolya; Tuboly, Gabor; Szucs, Maria; Birkas, Erika; Morvay, Zita; Benedek, Gyorgy; Horvath, Gyongyi

2011-07-01

Ketamine treatments and social isolation of rats reflect certain features of schizophrenia, among them altered pain sensitivity. To study the underlying mechanisms of these phenomena, rats were either housed individually or grouped for 33 days after weaning, and treated with either ketamine or saline for 14 days. After one month re-socialization, the urinary bladder capacity by ultrasound examination in the anesthetized animals, and changes of μ-opioid receptors by saturation binding experiments using a specific μ-opioid agonist [(3)H]DAMGO were determined. G-protein signaling was investigated in DAMGO-stimulated [(35)S]GTPγS functional assays. Ketamine treatment significantly decreased the bladder volume and isolation decreased the receptor density in cortical membranes. Among all groups, the only change in binding affinity was an increase induced by social isolation in the cortex. G-protein signaling was significantly decreased by either ketamine or social isolation in this tissue. Ketamine treatment, but not housing, significantly increased μ-opioid receptor densities in hippocampal membranes. Both ketamine and isolation increased the efficacy, while the potency of signaling was decreased by any treatment. Ketamine increased the receptor density and G-protein activation; while isolation decreased the efficacy of G-protein signaling in hippocampal membranes. The changes in the co-treated group were similar to those of the isolated animals in most tests. The distinct changes of opioid receptor functioning in different areas of the CNS may, at least partially, explain the augmented nociceptive threshold and morphine potency observed in these animals. Changes in the relative urinary bladder suggest a detrusor hyperreflexia, another sign of schizophrenia. Copyright © 2011 Elsevier B.V. All rights reserved.

Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries.

PubMed

Nuttall, S D; Krishnan, U V; Hattarki, M; De Gori, R; Irving, R A; Hudson, P J

2001-08-01

The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein. To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages. On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis. One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E. coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein. Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues. Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries.

Intrinsically-disordered N-termini in human parechovirus 1 capsid proteins bind encapsidated RNA.

PubMed

Shakeel, Shabih; Evans, James D; Hazelbaker, Mark; Kao, C Cheng; Vaughan, Robert C; Butcher, Sarah J

2018-04-11

Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantly-expressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins' N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0.

Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins.

PubMed

Valdivieso-Torres, Leonardo; Sarangi, Anindita; Whidby, Jillian; Marcotrigiano, Joseph; Roth, Monica J

2015-12-30

Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism are not understood. This study highlights the advantage of constrained peptides within receptor binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 envelopes that are essential for viral infection have been identified in this study and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F and W) and basic (K and H) residues critical for viral titer and suggest that AII, BV2, and CP internal cysteines bond together in distinct ways. Retargeted gammaretroviral particles have broad applications for therapeutic use. Although great advances have been achieved in identifying new Env-host cell receptor pairs, the rules for designing optimal Env libraries are still unclear. We have found that isolates with an additional pair of cysteines within the randomized region have the highest transduction efficiencies. This emphasizes the importance of considering cysteine pairs in the design of new libraries. Furthermore, our data clearly indicate that these cysteines are essential for viral infectivity by presenting essential residues to the host cell receptor. These studies facilitate the screening of Env libraries for functional entry into target cells, allowing the identification of novel gammaretroviral Envs targeting alternative host cell receptors for gene and protein delivery. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Dissecting protein:protein interactions between transcription factors with an RNA aptamer.

PubMed Central

Tian, Y; Adya, N; Wagner, S; Giam, C Z; Green, M R; Ellington, A D

1995-01-01

Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies. PMID:7489503

Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

Treesearch

Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

2001-01-01

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

Plasma biomarker discovery in preeclampsia using a novel differential isolation technology for circulating extracellular vesicles.

PubMed

Tan, Kok Hian; Tan, Soon Sim; Sze, Siu Kwan; Lee, Wai Kheong Ryan; Ng, Mor Jack; Lim, Sai Kiang

2014-10-01

To circumvent the complex protein milieu of plasma and discover robust predictive biomarkers for preeclampsia (PE), we investigate if phospholipid-binding ligands can reduce the milieu complexity by extracting plasma extracellular vesicles for biomarker discovery. Cholera toxin B chain (CTB) and annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine were used to isolate extracellular vesicles from plasma of PE patients and healthy pregnant women. The proteins in the vesicles were identified using enzyme-linked immunosorbent assay, antibody array, and mass spectrometry. CTB and AV were found to bind 2 distinct groups of extracellular vesicles. Antibody array and enzyme-linked immunosorbent assay revealed that PE patients had elevated levels of CD105, interleukin-6, placental growth factor, tissue inhibitor of metallopeptidase 1, and atrial natriuretic peptide in cholera toxin B- but not AV-vesicles, and elevated levels of plasminogen activator inhibitor-1, pro-calcitonin, S100b, tumor growth factor β, vascular endothelial growth factor receptor 1, brain natriuretic peptide, and placental growth factor in both cholera toxin B- and AV-vesicles. CD9 level was elevated in cholera toxin B-vesicles but reduced in AV vesicles of PE patients. Proteome analysis revealed that in cholera toxin B-vesicles, 87 and 222 proteins were present only in PE patients and healthy pregnant women respectively while in AV-vesicles, 104 and 157 proteins were present only in PE and healthy pregnant women, respectively. This study demonstrated for the first time that CTB and AV bind unique extracellular vesicles, and their protein cargo reflects the disease state of the patient. The successful use of these 2 ligands to isolate circulating plasma extracellular vesicles for biomarker discovery in PE represents a novel technology for biomarker discovery that can be applied to other specialties. Copyright © 2014 Elsevier Inc. All rights reserved.

Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain.

PubMed Central

Stevenson, S C; Rollence, M; White, B; Weaver, L; McClelland, A

1995-01-01

The adenovirus fiber protein is responsible for attachment of the virion to cell surface receptors. The identity of the cellular receptor which mediates binding is unknown, although there is evidence suggesting that two distinct adenovirus receptors interact with the group C (adenovirus type 5 [Ad5]) and the group B (Ad3) adenoviruses. In order to define the determinants of adenovirus receptor specificity, we have carried out a series of competition binding experiments using recombinant native fiber polypeptides from Ad5 and Ad3 and chimeric fiber proteins in which the head domains of Ad5 and Ad3 were exchanged. Specific binding of fiber to HeLa cell receptors was assessed with radiolabeled protein synthesized in vitro, and by competition analysis with baculovirus-expressed fiber protein. Fiber produced in vitro was found as both monomer and trimer, but only the assembled trimers had receptor binding activity. Competition data support the conclusion that Ad5 and Ad3 interact with different cellular receptors. The Ad5 receptor distribution on several cell lines was assessed with a fiber binding flow cytometric assay. HeLa cells were found to express high levels of receptor, while CHO and human diploid fibroblasts did not. A chimeric fiber containing the Ad5 fiber head domain blocked the binding of Ad5 fiber but not Ad3 fiber. Similarly, a chimeric fiber containing the Ad3 fiber head blocked the binding of labeled Ad3 fiber but not Ad5 fiber. In addition, the isolated Ad3 fiber head domain competed effectively with labeled Ad3 fiber for binding to HeLa cell receptors. These results demonstrate that the determinants of receptor binding are located in the head domain of the fiber and that the isolated head domain is capable of trimerization and binding to cellular receptors. Our results also show that it is possible to change the receptor specificity of the fiber protein by manipulation of sequences contained in the head domain. Modification or replacement of the fiber head domain with novel ligands may permit adenovirus vectors with new receptor specificities which could be useful for targeted gene delivery in vivo to be engineered. PMID:7707507

Toxic compound, anti-nutritional factors and functional properties of protein isolated from detoxified Jatropha curcas seed cake.

PubMed

Saetae, Donlaporn; Suntornsuk, Worapot

2010-12-28

Jatropha curcas is a multipurpose tree, which has potential as an alternative source for biodiesel. All of its parts can also be used for human food, animal feed, fertilizer, fuel and traditional medicine. J. curcas seed cake is a low-value by-product obtained from biodiesel production. The seed cake, however, has a high amount of protein, with the presence of a main toxic compound: phorbol esters as well as anti-nutritional factors: trypsin inhibitors, phytic acid, lectin and saponin. The objective of this work was to detoxify J. curcas seed cake and study the toxin, anti-nutritional factors and also functional properties of the protein isolated from the detoxified seed cake. The yield of protein isolate was approximately 70.9%. The protein isolate was obtained without a detectable level of phorbol esters. The solubility of the protein isolate was maximal at pH 12.0 and minimal at pH 4.0. The water and oil binding capacities of the protein isolate were 1.76 g water/g protein and 1.07 mL oil/g protein, respectively. The foam capacity and stability, including emulsion activity and stability of protein isolate, had higher values in a range of basic pHs, while foam and emulsion stabilities decreased with increasing time. The results suggest that the detoxified J. curcas seed cake has potential to be exploited as a novel source of functional protein for food applications.

Toxic Compound, Anti-Nutritional Factors and Functional Properties of Protein Isolated from Detoxified Jatropha curcas Seed Cake

PubMed Central

Saetae, Donlaporn; Suntornsuk, Worapot

2011-01-01

Jatropha curcas is a multipurpose tree, which has potential as an alternative source for biodiesel. All of its parts can also be used for human food, animal feed, fertilizer, fuel and traditional medicine. J. curcas seed cake is a low-value by-product obtained from biodiesel production. The seed cake, however, has a high amount of protein, with the presence of a main toxic compound: phorbol esters as well as anti-nutritional factors: trypsin inhibitors, phytic acid, lectin and saponin. The objective of this work was to detoxify J. curcas seed cake and study the toxin, anti-nutritional factors and also functional properties of the protein isolated from the detoxified seed cake. The yield of protein isolate was approximately 70.9%. The protein isolate was obtained without a detectable level of phorbol esters. The solubility of the protein isolate was maximal at pH 12.0 and minimal at pH 4.0. The water and oil binding capacities of the protein isolate were 1.76 g water/g protein and 1.07 mL oil/g protein, respectively. The foam capacity and stability, including emulsion activity and stability of protein isolate, had higher values in a range of basic pHs, while foam and emulsion stabilities decreased with increasing time. The results suggest that the detoxified J. curcas seed cake has potential to be exploited as a novel source of functional protein for food applications. PMID:21339978

Isolation and characterization of a Ca/sup 2 +/ carrier candidate from calf heart inner mitochondrial membrane

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

Jeng, A.Y.

1979-01-01

A protein was isolated from calf heart inner mitochondrial membrane with the aid of an electron paramagnetic resonance assay based on the relative binding properties of Ca/sup 2 +/, Mn/sup 2 +/, and Mg/sup 2 +/ to the protein. Partial delipidation of the protein was performed by using either the organic solvent extraction procedure or the silicic acid column chromatography. Control experiments indicated that the Ca/sup 2 +/ transport properties of the isolated protein were not due to the contaminating phospholipids. A complete delipidation procedure was developd by using Sephadex LH-20 column chromatography. Further characterization of the physical and chemicalmore » properties of the delipidated protein showed that delipidated protein becomes more hydrophobic in the presence of Ca/sup 2 +/ and alkaline pH in the organic solvent extraction experiments. Two possible models of calciphorin-mediated Ca/sup 2 +/ transport in mitochondria are proposed. (PCS)« less

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification.

PubMed

Bredow, Melissa; Tomalty, Heather E; Walker, Virginia K

2017-05-05

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a 'splat assay', which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called 'ice-affinity purification'. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

Influence of extraction pH on the foaming, emulsification, oil-binding and visco-elastic properties of marama protein.

PubMed

Gulzar, Muhammad; Taylor, John Rn; Minnaar, Amanda

2017-11-01

Marama bean protein, as extracted previously at pH 8, forms a viscous, adhesive and extensible dough. To obtain a protein isolate with optimum functional properties, protein extraction under slightly acidic conditions (pH 6) was investigated. Two-dimensional electrophoresis showed that pH 6 extracted marama protein lacked some basic 11S legumin polypeptides, present in pH 8 extracted protein. However, it additionally contained acidic high molecular weight polypeptides (∼180 kDa), which were disulfide crosslinked into larger proteins. pH 6 extracted marama proteins had similar emulsification properties to soy protein isolate and several times higher foaming capacity than pH 8 extracted protein, egg white and soy protein isolate. pH 6 extracted protein dough was more elastic than pH 8 extracted protein, approaching the elasticity of wheat gluten. Marama protein extracted at pH 6 has excellent food-type functional properties, probably because it lacks some 11S polypeptides but has additional high molecular weight proteins. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

The Molecular Chaperone TRiC/CCT Binds to the Trp-Asp 40 (WD40) Repeat Protein WDR68 and Promotes Its Folding, Protein Kinase DYRK1A Binding, and Nuclear Accumulation*

PubMed Central

Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

2014-01-01

Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68. PMID:25342745

A Major Binding Protein for Leukemia Inhibitory Factor in Normal Mouse Serum: Identification as a Soluble Form of the Cellular Receptor

NASA Astrophysics Data System (ADS)

Layton, Meredith J.; Cross, Bronwyn A.; Metcalf, Donald; Ward, Larry D.; Simpson, Richard J.; Nicola, Nicos A.

1992-09-01

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (K_d = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the α chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 μg/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

A Non-Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH-Modulation of Protein-Nucleic Acid Interfaces.

PubMed

Cruz-Gallardo, Isabel; Del Conte, Rebecca; Velázquez-Campoy, Adrián; García-Mauriño, Sofía M; Díaz-Moreno, Irene

2015-05-11

A useful (2) J(N-H) coupling-based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA-binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T-cell intracellular antigen-1 (TIA-1) protein. The pKa values of the His96 ionizable groups were substantially higher in the complexes with short U-rich RNA and T-rich DNA oligonucleotides than those of the isolated TIA-1 RRM2. Herein, the methodology applied to determine changes in pKa of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA-binding proteins that shuttle among different cellular compartments. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RIPiT-Seq: A high-throughput approach for footprinting RNA:protein complexes

PubMed Central

Singh, Guramrit; Ricci, Emiliano P.; Moore, Melissa J.

2013-01-01

Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications; the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. Further, among current high-throughput approaches, RIPiT has the unique capacity to differentiate binding sites of RNPs with overlapping protein composition. It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds. PMID:24096052

Scarabaecin, a novel cysteine-containing antifungal peptide from the rhinoceros beetle, Oryctes rhinoceros.

PubMed

Tomie, Tetsuya; Ishibashi, Jun; Furukawa, Seiichi; Kobayashi, Satoe; Sawahata, Ryoko; Asaoka, Ai; Tagawa, Michito; Yamakawa, Minoru

2003-07-25

A novel antifungal peptide, scarabaecin (4080Da), was isolated from the coconut rhinoceros beetle, Oryctes rhinoceros. Scarabaecin cDNA was cloned by reverse transcriptase-polymerase chain reactions (RT-PCR) using a primer based on the N-terminal amino acid sequence. The amino acid sequence deduced from scarabaecin cDNA showed no significant similarity to those of reported proteins. Chemically synthesized scarabaecin indicated antifungal activity against phytopathogenic fungi such as Pyricularia oryzae, Rhizoctonia solani, and Botrytis cinerea, but not against phytopathogenic bacteria. It showed weak activity against Bauberia bassiana, an insect pathogenic fungus, and Staphylococcus aureus, a pathogenic bacterium. Scarabaecin showed chitin binding property and its K(d) was 1.315 microM. A comparison of putative chitin-binding domains among scarabaecin, invertebrate, and plant chitin-binding proteins suggests that scarabaecin is a new member of chitin-binding antimicrobial proteins.

Assessment of Cpa, Scl1 and Scl2 in clinical group A streptococcus isolates and patients from north India: an evaluation of the host pathogen interaction.

PubMed

Chaudhary, Priyanka; Kumar, Rajesh; Sagar, Vivek; Sarkar, Subendu; Singh, Rupneet; Ghosh, Sujata; Singh, Surjit; Chakraborti, Anuradha

2018-01-01

Group A streptococcus (GAS) infection remains a major concern due to multiple diseases including pharyngitis, impetigo, acute rheumatic fever (ARF) and rheumatic heart disease (RHD). It uses different adhesins and virulence factors like Cpa (collagen binding protein) and Scl (collagen-like protein) in its pathogenicity. Scl having similarities with human collagen may contribute to inducing autoimmunity in the host. Here we assessed gene expression, antibody titer of Cpa, Scl1 and Scl2 in both clinical GAS isolates (n = 45) and blood (n = 45) obtained from pharyngitis, ARF (acute rheumatic fever) and RHD respectively. Skin isolates (n = 30) were obtained from impetigo patients. The study revealed a total of 27 GAS emm types. Frequency of cpa, scl1, scl2 was high in ARF isolates. The antibody titer of these proteins was high in all isolates, and also in patients with pharyngitis and ARF. All isolates showed high binding affinity toward collagen I and IV, which further indicates a potential host pathogen interaction. Our study reflects a strong association of Cpa and Scls in early and post-GAS pathogenicity. However, the increased antibody titer of Scl1 and Scl2 during ARF may be attributed to a cogent immune response in the host. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Techniques for quantitative LC-MS/MS analysis of protein therapeutics: advances in enzyme digestion and immunocapture.

PubMed

Fung, Eliza N; Bryan, Peter; Kozhich, Alexander

2016-04-01

LC-MS/MS has been investigated to quantify protein therapeutics in biological matrices. The protein therapeutics is digested by an enzyme to generate surrogate peptide(s) before LC-MS/MS analysis. One challenge is isolating protein therapeutics in the presence of large number of endogenous proteins in biological matrices. Immunocapture, in which a capture agent is used to preferentially bind the protein therapeutics over other proteins, is gaining traction. The protein therapeutics is eluted for digestion and LC-MS/MS analysis. One area of tremendous potential for immunocapture-LC-MS/MS is to obtain quantitative data where ligand-binding assay alone is not sufficient, for example, quantitation of antidrug antibody complexes. Herein, we present an overview of recent advance in enzyme digestion and immunocapture applicable to protein quantitation.

Virulence Gene Pool Detected in Bovine Group C Streptococcus dysgalactiae subsp. dysgalactiae Isolates by Use of a Group A S. pyogenes Virulence Microarray ▿

PubMed Central

Rato, Márcia G.; Nerlich, Andreas; Bergmann, René; Bexiga, Ricardo; Nunes, Sandro F.; Vilela, Cristina L.; Santos-Sanches, Ilda; Chhatwal, Gursharan S.

2011-01-01

A custom-designed microarray containing 220 virulence genes of Streptococcus pyogenes (group A Streptococcus [GAS]) was used to test group C Streptococcus dysgalactiae subsp. dysgalactiae (GCS) field strains causing bovine mastitis and group C or group G Streptococcus dysgalactiae subsp. equisimilis (GCS/GGS) isolates from human infections, with the latter being used for comparative purposes, for the presence of virulence genes. All bovine and all human isolates carried a fraction of the 220 genes (23% and 39%, respectively). The virulence genes encoding streptolysin S, glyceraldehyde-3-phosphate dehydrogenase, the plasminogen-binding M-like protein PAM, and the collagen-like protein SclB were detected in the majority of both bovine and human isolates (94 to 100%). Virulence factors, usually carried by human beta-hemolytic streptococcal pathogens, such as streptokinase, laminin-binding protein, and the C5a peptidase precursor, were detected in all human isolates but not in bovine isolates. Additionally, GAS bacteriophage-associated virulence genes encoding superantigens, DNase, and/or streptodornase were detected in bovine isolates (72%) but not in the human isolates. Determinants located in non-bacteriophage-related mobile elements, such as the gene encoding R28, were detected in all bovine and human isolates. Several virulence genes, including genes of bacteriophage origin, were shown to be expressed by reverse transcriptase PCR (RT-PCR). Phylogenetic analysis of superantigen gene sequences revealed a high level (>98%) of identity among genes of bovine GCS, of the horse pathogen Streptococcus equi subsp. equi, and of the human pathogen GAS. Our findings indicate that alpha-hemolytic bovine GCS, an important mastitis pathogen and considered to be a nonhuman pathogen, carries important virulence factors responsible for virulence and pathogenesis in humans. PMID:21525223

Nonhemolytic Streptococcus pyogenes Isolates That Lack Large Regions of the sag Operon Mediating Streptolysin S Production▿

PubMed Central

Yoshino, Miho; Murayama, Somay Y.; Sunaoshi, Katsuhiko; Wajima, Takeaki; Takahashi, Miki; Masaki, Junko; Kurokawa, Iku; Ubukata, Kimiko

2010-01-01

Among nonhemolytic Streptococcus pyogenes (group A streptococcus) strains (n = 9) isolated from patients with pharyngitis or acute otitis media, we identified three deletions in the region from the epf gene, encoding the extracellular matrix binding protein, to the sag operon, mediating streptolysin S production. PMID:20018818

Highly Pathogenic Influenza A(H5Nx) Viruses with Altered H5 Receptor-Binding Specificity

PubMed Central

Guo, Hongbo; de Vries, Erik; McBride, Ryan; Dekkers, Jojanneke; Peng, Wenjie; Bouwman, Kim M.; Nycholat, Corwin; Verheije, M. Helene; Paulson, James C.; van Kuppeveld, Frank J.M.

2017-01-01

Emergence and intercontinental spread of highly pathogenic avian influenza A(H5Nx) virus clade 2.3.4.4 is unprecedented. H5N8 and H5N2 viruses have caused major economic losses in the poultry industry in Europe and North America, and lethal human infections with H5N6 virus have occurred in Asia. Knowledge of the evolution of receptor-binding specificity of these viruses, which might affect host range, is urgently needed. We report that emergence of these viruses is accompanied by a change in receptor-binding specificity. In contrast to ancestral clade 2.3.4 H5 proteins, novel clade 2.3.4.4 H5 proteins bind to fucosylated sialosides because of substitutions K222Q and S227R, which are unique for highly pathogenic influenza virus H5 proteins. North American clade 2.3.4.4 virus isolates have retained only the K222Q substitution but still bind fucosylated sialosides. Altered receptor-binding specificity of virus clade 2.3.4.4 H5 proteins might have contributed to emergence and spread of H5Nx viruses. PMID:27869615

Cadmium is deposited in the gut content of larvae of the beetle Tenebrio molitor and involves a Cd-binding protein of the low cysteine type.

PubMed

Pedersen, S A; Kristiansen, E; Andersen, R A; Zachariassen, K E

2008-09-01

Binding of cadmium (Cd) to metallothionein (MT) and non-MT proteins with low contents of cysteine has been observed in terrestrial arthropods. We recently isolated a Cd-binding protein with no cysteine that was induced in Cd-exposed larvae of the beetle Tenebrio molitor. In this study we have examined the molecular distribution of Cd within extracts of different tissues and compartments of Cd-exposed T. molitor larvae. A Cd-peak consistent with the low cysteine Cd-binding protein was induced within the gut content where it could be detected after 4-8 days of exposure. Examination of gut wall tissue revealed no increase in Cd-binding capacity, indicating that no accumulation of MTs was taking place in this tissue. Incorporation of Cd in the gut wall tissue stabilized after 8 days of Cd-exposure at a rather low level compared to the other organs. There was a statistical trend towards Cd being incorporated in the gut content in a manner that was disproportionally high compared to the amount of Cd in the gut wall tissue. The possible role of the low cysteine Cd-binding protein in reducing the uptake of Cd in the tissues is discussed.

Interactions of the chemotaxis signal protein CheY with bacterial flagellar motors visualized by evanescent wave microscopy.

PubMed

Khan, S; Pierce, D; Vale, R D

The chemotaxis signal protein CheY of enteric bacteria shuttles between transmembrane methyl-accepting chemotaxis protein (MCP) receptor complexes and flagellar basal bodies [1]. The basal body C-rings, composed of the FliM, FliG and FliN proteins, form the rotor of the flagellar motor [2]. Phosphorylated CheY binds to isolated FliM [3] and may also interact with FliG [4], but its binding to basal bodies has not been measured. Using the chemorepellent acetate to phosphorylate and acetylate CheY [5], we have measured the covalent-modification-dependent binding of a green fluorescent protein-CheY fusion (GFP-CheY) to motor assemblies in bacteria lacking MCP complexes by evanescent wave microscopy [6]. At acetate concentrations that cause solely clockwise rotation, GFP-CheY molecules bound to native basal bodies or to overproduced rotor complexes with a stoichiometry comparable to the number of C-ring subunits. GFP-CheY did not bind to rotors lacking FIiM/FliN, showing that these subunits are essential for the association. This assay provides a new means of monitoring protein-protein interactions in signal transduction pathways in living cells.

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

PubMed

Morea, Edna G O; Viviescas, Maria Alejandra; Fernandes, Carlos A H; Matioli, Fabio F; Lira, Cristina B B; Fernandez, Maribel F; Moraes, Barbara S; da Silva, Marcelo S; Storti, Camila B; Fontes, Marcos R M; Cano, Maria Isabel N

2017-11-01

Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. LCalA is the first reported calmodulin that binds in vivo telomeric DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain.

PubMed

Wu, R; Wilton, R; Cuff, M E; Endres, M; Babnigg, G; Edirisinghe, J N; Henry, C S; Joachimiak, A; Schiffer, M; Pokkuluri, P R

2017-04-01

We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from Lake Retba, in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously, and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport. © 2017 The Protein Society.

Membrane proteins from the cyanobacterium Synechocystis sp. PCC 6803 interacting with thioredoxin.

PubMed

Mata-Cabana, Alejandro; Florencio, Francisco J; Lindahl, Marika

2007-11-01

Cysteine dithiol/disulphide exchange forms the molecular basis for regulation of a wide variety of enzymatic activities and for transduction of cellular signals. Thus, the search for proteins with reactive, accessible cysteines is expected to contribute to the unravelling of new molecular mechanisms for enzyme regulation and signal transduction. Several methods have been designed for this purpose taking advantage of the interactions between thioredoxins and their protein substrates. Thioredoxins comprise a family of redox-active enzymes, which catalyse reduction of protein disulphides and sulphenic acids. Due to the inherent practical difficulties associated with studies of membrane proteins these have been largely overlooked in the many proteomic studies of thioredoxin-interacting proteins. In the present work, we have developed a procedure to isolate membrane proteins interacting with thioredoxin by binding in situ to a monocysteinic His-tagged thioredoxin added directly to the intact membranes. Following fractionation and solubilisation of the membranes, thioredoxin target proteins were isolated by Ni-affinity chromatography and 2-DE SDS-PAGE under nonreducing/reducing conditions. Applying this method to total membranes, including thylakoid and plasma membranes, from the cyanobacterium Synechocystis sp. PCC 6803 we have identified 50 thioredoxin-interacting proteins. Among the 38 newly identified thioredoxin targets are the ATP-binding subunits of several transporters and members of the AAA-family of ATPases.

Structure of pleiotrophin- and hepatocyte growth factor-binding sulfated hexasaccharide determined by biochemical and computational approaches.

PubMed

Li, Fuchuan; Nandini, Chilkunda D; Hattori, Tomohide; Bao, Xingfeng; Murayama, Daisuke; Nakamura, Toshikazu; Fukushima, Nobuhiro; Sugahara, Kazuyuki

2010-09-03

Endogenous pleiotrophin and hepatocyte growth factor (HGF) mediate the neurite outgrowth-promoting activity of chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains isolated from embryonic pig brain. CS/DS hybrid chains isolated from shark skin have a different disaccharide composition, but also display these activities. In this study, pleiotrophin- and HGF-binding domains in shark skin CS/DS were investigated. A high affinity CS/DS fraction was isolated using a pleiotrophin-immobilized column. It showed marked neurite outgrowth-promoting activity and strong inhibitory activity against the binding of pleiotrophin to immobilized CS/DS chains from embryonic pig brain. The inhibitory activity was abolished by chondroitinase ABC or B, and partially reduced by chondroitinase AC-I. A pentasulfated hexasaccharide with a novel structure was isolated from the chondroitinase AC-I digest using pleiotrophin affinity and anion exchange chromatographies. It displayed a potent inhibitory effect on the binding of HGF to immobilized shark skin CS/DS chains, suggesting that the pleiotrophin- and HGF-binding domains at least partially overlap in the CS/DS chains involved in the neuritogenic activity. Computational chemistry using molecular modeling and calculations of the electrostatic potential of the hexasaccharide and two pleiotrophin-binding octasaccharides previously isolated from CS/DS hybrid chains of embryonic pig brain identified an electronegative zone potentially involved in the molecular recognition of the oligosaccharides by pleiotrophin. Homology modeling of pleiotrophin based on a related midkine protein structure predicted the binding pocket of pleiotrophin for the oligosaccharides and provided new insights into the molecular mechanism of the interactions between the oligosaccharides and pleiotrophin.

Identification of a 27.8 kDa protein from flounder gill cells involved in lymphocystis disease virus binding and infection.

PubMed

Wang, Mu; Sheng, Xiu-Zhen; Xing, Jing; Tang, Xiao-Qian; Zhan, Wen-Bin

2011-03-16

In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDV-binding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and beta-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells.

Screening a yeast promoter library leads to the isolation of the RP29/L32 and SNR17B/RPL37A divergent promoters and the discovery of a gene encoding ribosomal protein L37.

PubMed

Santangelo, G M; Tornow, J; McLaughlin, C S; Moldave, K

1991-08-30

Two promoters (A7 and A23), isolated at random from the Saccharomyces cerevisiae genome by virtue of their capacity to activate transcription, are identical to known intergenic bidirectional promoters. Sequence analysis of the genomic DNA adjacent to the A7 promoter identified a split gene encoding ribosomal (r) protein L37, which is homologous to the tRNA-binding r-proteins, L35a (from human and rat) and L32 (from frogs).

Fragments of the V1/V2 domain of HIV-1 glycoprotein 120 engineered for improved binding to the broadly neutralizing PG9 antibody.

PubMed

Morales, Javier F; Yu, Bin; Perez, Gerardo; Mesa, Kathryn A; Alexander, David L; Berman, Phillip W

2016-09-01

The V1/V2 domain of the HIV-1 envelope protein gp120 possesses two important epitopes: a glycan-dependent epitope recognized by the prototypic broadly neutralizing monoclonal antibody (bN-mAb), PG9, as well as an epitope recognized by non-neutralizing antibodies that has been associated with protection from HIV infection in the RV144 HIV vaccine trial. Because both of these epitopes are poorly immunogenic in the context of full length envelope proteins, immunization with properly folded and glycosylated fragments (scaffolds) represents a potential way to enhance the immune response to these specific epitopes. Previous studies showed that V1/V2 domain scaffolds could be produced from a few selected isolates, but not from many of the isolates that would be advantageous in a multivalent vaccine. In this paper, we used a protein engineering approach to improve the conformational stability and antibody binding activity of V1/V2 domain scaffolds from multiple diverse isolates, including several that were initially unable to bind the prototypic PG9 bN-mAb. Significantly, this effort required replicating both the correct glycan structure as well as the β-sheet structure required for PG9 binding. Although scaffolds incorporating the glycans required for PG9 binding (e.g., mannose-5) can be produced using glycosylation inhibitors (e.g., swainsonine), or mutant cell lines (e.g. GnTI(-) 293 HEK), these are not practical for biopharmaceutical production of proteins intended for clinical trials. In this report, we describe engineered glycopeptide scaffolds from three different clades of HIV-1 that bind PG9 with high affinity when expressed in a wildtype cell line suitable for biopharmaceutical production. The mutations that improved PG9 binding to scaffolds produced in normal cells included amino acid positions outside of the antibody contact region designed to stabilize the β-sheet and turn structures. The scaffolds produced address three major problems in HIV vaccine development: (1) improving antibody responses to poorly immunogenic epitopes in the V1/V2 domain; (2) eliminating antibody responses to highly immunogenic (decoy) epitopes outside the V1/V2 domain; and (3) enabling the production of V1/V2 scaffolds in a cell line suitable for biopharmaceutical production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phosducin-like protein: an ethanol-responsive potential modulator of guanine nucleotide-binding protein function.

PubMed

Miles, M F; Barhite, S; Sganga, M; Elliott, M

1993-11-15

Acute and chronic exposure to ethanol produces specific changes in several signal transduction cascades. Such alterations in signaling are thought to be a crucial aspect of the central nervous system's adaptive response, which occurs with chronic exposure to ethanol. We have recently identified and isolated several genes whose expression is specifically induced by ethanol in neural cell cultures. The product of one of these genes has extensive sequence homology to phosducin, a phosphoprotein expressed in retina and pineal gland that modulates trimeric guanine nucleotide-binding protein (G protein) function by binding to G-protein beta gamma subunits. We identified from a rat brain cDNA library an isolate encoding the phosducin-like protein (PhLP), which has 41% identity and 65% amino acid homology to phosducin. PhLP cDNA is expressed in all tissues screened by RNA blot-hybridization analysis and shows marked evolutionary conservation on Southern hybridization. We have identified four forms of PhLP cDNA varying only in their 5' ends, probably due to alternative splicing. This 5'-end variation generates two predicted forms of PhLP protein that differ by 79 aa at the NH2 terminus. Treatment of NG108-15 cells for 24 hr with concentrations of ethanol seen in actively drinking alcoholics (25-100 mM) causes up to a 3-fold increase in PhLP mRNA levels. Induction of PhLP by ethanol could account for at least some of the widespread alterations in signal transduction and G-protein function that are known to occur with chronic exposure to ethanol.

Incorporation of deoxyribonucleotides and ribonucleotides by a dNTP-binding cleft mutated reverse transcriptase in hepatitis B virus core particles

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

Kim, Hee-Young; Kim, Hye-Young; Jung, Jaesung

2008-01-05

Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22-30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate {sup 32}P-ribonucleotides, but not HBV coremore » particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems.« less

A model for regulation by SynGAP-α1 of binding of synaptic proteins to PDZ-domain 'Slots' in the postsynaptic density

PubMed Central

Walkup, Ward G; Mastro, Tara L; Schenker, Leslie T; Vielmetter, Jost; Hu, Rebecca; Iancu, Ariella; Reghunathan, Meera; Bannon, Barry Dylan; Kennedy, Mary B

2016-01-01

SynGAP is a Ras/Rap GTPase-activating protein (GAP) that is a major constituent of postsynaptic densities (PSDs) from mammalian forebrain. Its α1 isoform binds to all three PDZ (PSD-95, Discs-large, ZO-1) domains of PSD-95, the principal PSD scaffold, and can occupy as many as 15% of these PDZ domains. We present evidence that synGAP-α1 regulates the composition of the PSD by restricting binding to the PDZ domains of PSD-95. We show that phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Polo-like kinase-2 (PLK2) decreases its affinity for the PDZ domains by several fold, which would free PDZ domains for occupancy by other proteins. Finally, we show that three critical postsynaptic signaling proteins that bind to the PDZ domains of PSD-95 are present in higher concentration in PSDs isolated from mice with a heterozygous deletion of synGAP. DOI: http://dx.doi.org/10.7554/eLife.16813.001 PMID:27623146

DNA wrapping and distortion by an oligomeric homeodomain protein.

PubMed

Williams, Hannah; Jayaraman, Padma-Sheela; Gaston, Kevin

2008-10-31

Many transcription factors alter DNA or chromatin structure. Changes in chromatin structure are often brought about by the recruitment of chromatin-binding proteins, chromatin-modifying proteins, or other transcription co-activator or co-repressor proteins. However, some transcription factors form oligomeric assemblies that may themselves induce changes in DNA conformation and chromatin structure. The proline-rich homeodomain (PRH/Hex) protein is a transcription factor that regulates cell differentiation and cell proliferation, and has multiple roles in embryonic development. Earlier, we showed that PRH can repress transcription by multiple mechanisms, including the recruitment of co-repressor proteins belonging to the TLE family of chromatin-binding proteins. Our in vivo crosslinking studies have shown that PRH forms oligomeric complexes in cells and a variety of biophysical techniques suggest that the protein forms octamers. However, as yet we have little knowledge of the role played by PRH oligomerisation in the regulation of promoter activity or of the architecture of promoters that are regulated directly by PRH in cells. Here, we compare the binding of PRH and the isolated PRH homeodomain to DNA fragments with single and multiple PRH sites, using gel retardation assays and DNase I and chemical footprinting. We show that the PRH oligomer binds to multiple sites within the human Goosecoid promoter with high affinity and that the binding of PRH brings about DNA distortion. We suggest that PRH octamers wrap DNA in order to bring about transcriptional repression.

Isolation, Characterization and Lipid-Binding Properties of the Recalcitrant FtsA Division Protein from Escherichia coli

PubMed Central

Zorrilla, Silvia; Reija, Belén; Alfonso, Carlos; Mingorance, Jesús; Rivas, Germán; Jiménez, Mercedes

2012-01-01

We have obtained milligram amounts of highly pure Escherichia coli division protein FtsA from inclusion bodies with an optimized purification method that, by overcoming the reluctance of FtsA to be purified, surmounts a bottleneck for the analysis of the molecular basis of FtsA function. Purified FtsA is folded, mostly monomeric and interacts with lipids. The apparent affinity of FtsA binding to the inner membrane is ten-fold higher than to phospholipids, suggesting that inner membrane proteins could modulate FtsA-membrane interactions. Binding of FtsA to lipids and membranes is insensitive to ionic strength, indicating that a net contribution of hydrophobic interactions is involved in the association of FtsA to lipid/membrane structures. PMID:22761913

The LIM Protein Zyxin Binds CARP-1 and Promotes Apoptosis

PubMed Central

Hervy, Martial; Hoffman, Laura M.; Jensen, Christopher C.; Smith, Mark; Beckerle, Mary C.

2010-01-01

Zyxin is a dual-function LIM domain protein that regulates actin dynamics in response to mechanical stress and shuttles between focal adhesions and the cell nucleus. Here we show that zyxin contributes to UV-induced apoptosis. Exposure of wild-type fibroblasts to UV-C irradiation results in apoptotic cell death, whereas cells harboring a homozygous disruption of the zyxin gene display a statistically significant survival advantage. To gain insight into the molecular mechanism by which zyxin promotes apoptotic signaling, we expressed an affinity-tagged zyxin variant in zyxin-null cells and isolated zyxin-associated proteins from cell lysates under physiological conditions. A 130-kDa protein that was co-isolated with zyxin was identified by microsequence analysis as the Cell Cycle and Apoptosis Regulator Protein-1 (CARP-1). CARP-1 associates with the LIM region of zyxin. Zyxin lacking the CARP-1 binding region shows reduced proapoptotic activity in response to UV-C irradiation. We demonstrate that CARP-1 is a nuclear protein. Zyxin is modified by phosphorylation in cells exposed to UV-C irradiation, and nuclear accumulation of zyxin is induced by UV-C exposure. These findings highlight a novel mechanism for modulating the apoptotic response to UV irradiation. PMID:20852740

Screening, Characterization and In Vitro Evaluation of Probiotic Properties Among Lactic Acid Bacteria Through Comparative Analysis.

PubMed

Devi, Sundru Manjulata; Archer, Ann Catherine; Halami, Prakash M

2015-09-01

The present work aimed to identify probiotic bacteria from healthy human infant faecal and dairy samples. Subsequently, an assay was developed to evaluate the probiotic properties using comparative genetic approach for marker genes involved in adhesion to the intestinal epithelial layer. Several in vitro properties including tolerance to biological barriers (such as acid and bile), antimicrobial spectrum, resistance to simulated digestive fluids and cellular hydrophobicity were assessed. The potential probiotic cultures were rapidly characterized by morphological, physiological and molecular-based methods [such as RFLP, ITS, RAPD and (GTG)5]. Further analysis by 16S rDNA sequencing revealed that the selected isolates belong to Lactobacillus, Pediococcus and Enterococcus species. Two cultures of non-lactic, non-pathogenic Staphylococcus spp. were also isolated. The native isolates were able to survive under acidic, bile and simulated intestinal conditions. In addition, these cultures inhibited the growth of tested bacterial pathogens. Further, no correlation was observed between hydrophobicity and adhesion ability. Sequencing of probiotic marker genes such as bile salt hydrolase (bsh), fibronectin-binding protein (fbp) and mucin-binding protein (mub) for selected isolates revealed nucleotide variation. The probiotic binding domains were detected by several bioinformatic tools. The approach used in the study enabled the identification of potential probiotic domains responsible for adhesion of bacteria to intestinal epithelial layer, which may further assist in screening of novel probiotic bacteria. The rapid detection of binding domains will help in revealing the beneficial properties of the probiotic cultures. Further, studies will be performed to develop a novel probiotic product which will contribute in food and feed industry.

Identification of potential virulence factors of Cronobacter sakazakii isolates by comparative proteomic analysis.

PubMed

Ye, Yingwang; Li, Hui; Ling, Na; Han, Yongjia; Wu, Qingping; Xu, Xiaoke; Jiao, Rui; Gao, Jina

2016-01-18

Cronobacter is a group of important foodborne pathogens associated with neonatal meningitis, septicemia, and necrotizing enterocolitis. Among Cronobacter species, Cronobacter sakazakii is the most common species in terms of isolation frequency. However, the molecular basis involved in virulence differences among C. sakazakii isolates is still unknown. In this study, based on the determination of virulence differences of C. sakazakii G362 (virulent isolate) and L3101 (attenuated isolate) through intraperitoneal injection, histopathologic analysis (small intestine, kidney, and liver) further confirmed virulence differences. Thereafter, the potential virulence factors were determined using two-dimensional electrophoresis (2-DE) coupled with MALDI/TOP/TOF mass spectrometry. Among a total of 36 protein spots showing differential expression (fold change>1.2), we identified 31 different proteins, of which the expression abundance of 22 was increased in G362. These up-regulated proteins in G362 mainly contained DNA starvation/stationary phase protection protein Dps, OmpA, LuxS, ATP-dependent Clp protease ClpC, and ABC transporter substrate-binding proteins, which might be involved in virulence of C. sakazakii. This is the first report to determine the potential virulence factors of C. sakazakii isolates at the proteomic levels. Copyright © 2015 Elsevier B.V. All rights reserved.

In silico analysis of AHJD-like viruses, Staphylococcus aureus phages S24-1 and S13′, and study of phage S24-1 adsorption

PubMed Central

Uchiyama, Jumpei; Takemura-Uchiyama, Iyo; Kato, Shin-ichiro; Sato, Miho; Ujihara, Takako; Matsui, Hidehito; Hanaki, Hideaki; Daibata, Masanori; Matsuzaki, Shigenobu

2014-01-01

Staphylococcus aureus is a clinically important bacterium that is commensal in both humans and animals. Bacteriophage (phage) attachment to the host bacterial surface is an important process during phage infection, which involves interactions between phage receptor-binding proteins and host receptor molecules. However, little information is available on the receptor-binding protein of S. aureus phages. S. aureus virulent phages S24-1 and S13′ (family Podoviridae, genus AHJD-like viruses) were isolated from sewage. In the present study, we investigated the receptor-binding protein of AHJD-like viruses using phage S24-1. First, based on a comparative genomic analysis of phages S24-1 and S13′, open reading frame 16 (ORF16) of phage S24-1 was speculated to be the receptor-binding protein, which possibly determines the host range. Second, we demonstrated that this was the receptor-binding protein of phage S24-1. Third, our study suggested that wall teichoic acids in the cell walls of S. aureus are the main receptor molecules for ORF16 and phage S24-1. Finally, the C-terminal region of ORF16 may be essential for binding to S. aureus. These results strongly suggest that ORF16 of phage S24-1 and its homologs may be the receptor-binding proteins of AHJD-like viruses. PMID:24591378

Chemical biology of natural indolocarbazole products: 30 years since the discovery of staurosporine.

PubMed

Nakano, Hirofumi; Omura, Satoshi

2009-01-01

Staurosporine was discovered at the Kitasato Institute in 1977 while screening for microbial alkaloids using chemical detection methods. It was during the same era that protein kinase C was discovered and oncogene v-src was shown to have protein kinase activity. Staurosporine was first isolated from a culture of Actinomyces that originated in a soil sample collected in Mizusawa City, Japan. Thereafter, indolocarbazole compounds have been isolated from a variety of organisms. The biosynthesis of staurosporine and related indolocarbazoles was finally elucidated during the past decade through genetic and biochemical studies. Subsequently, several novel indolocarbazoles have been produced using combinatorial biosynthesis. In 1986, 9 years since its discovery, staurosporine and related indolocarbazoles were shown to be nanomolar inhibitors of protein kinases. They can thus be viewed as forerunners of today's crop of novel anticancer drugs. The finding led many pharmaceutical companies to search for selective protein kinase inhibitors by screening natural products and through chemical synthesis. In the 1990s, imatinib, a Bcr-Abl tyrosine kinase inhibitor, was synthesized and, following human clinical trials for chronic myelogenous leukemia, it was approved for use in the USA in 2001. In 1992, mammalian topoisomerases were shown to be targets for indolocarbazoles. This opened up new possibilities in that indolocarbazole compounds could selectively interact with ATP-binding sites of not only protein kinases but also other proteins that had slight differences in ATP-binding sites. ABCG2, an ATP-binding cassette transporter, was recently identified as an important new target for indolocarbazoles.

PknB remains an essential and a conserved target for drug development in susceptible and MDR strains of M. Tuberculosis.

PubMed

Gupta, Anamika; Pal, Sudhir K; Pandey, Divya; Fakir, Najneen A; Rathod, Sunita; Sinha, Dhiraj; SivaKumar, S; Sinha, Pallavi; Periera, Mycal; Balgam, Shilpa; Sekar, Gomathi; UmaDevi, K R; Anupurba, Shampa; Nema, Vijay

2017-08-18

The Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India. A total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations. The sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding. Hence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study says that it has no significant effect on the binding and hence the activity of the protein.

Polyamine binding to proteins in oat and Petunia protoplasts

NASA Technical Reports Server (NTRS)

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

1989-01-01

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

Polyamine binding to proteins in oat and Petunia protoplasts.

PubMed

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

1989-01-01

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

Cooperative DNA binding of heterologous proteins: Evidence for contact between the cyclic AMP receptor protein and RNA polymerase

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

Ren, Y.L.; Garges, S.; Adhya, S.

1988-06-01

Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP. One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP. CRP*598 canmore » activate lacP{sup +}-directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection (footprinting) indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the ({sup 32}P)lacP{sup +} fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription. Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding.« less

Identification of Actin-Binding Proteins from Maize Pollen

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

Staiger, C.J.

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

Texturized dairy proteins.

PubMed

Onwulata, Charles I; Phillips, John G; Tunick, Michael H; Qi, Phoebi X; Cooke, Peter H

2010-03-01

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 protein concentrate (WPC), and whey protein isolate (WPI) were modified using a twin-screw extruder at melt temperatures of 50, 75, and 100 degrees C, and moistures ranging from 20 to 70 wt%. Viscoelasticity and solubility measurements showed that extrusion temperature was a more significant (P < 0.05) change factor than moisture content. The degree of texturization, or change in protein state, was characterized by solubility (R(2)= 0.98). The consistency of the extruded dairy protein ranged from rigid (2500 N) to soft (2.7 N). Extruding at or above 75 degrees C resulted in increased peak force for WPC (138 to 2500 N) and WPI (2.7 to 147.1 N). NDM was marginally texturized; the presence of lactose interfered with its texturization. WPI products extruded at 50 degrees C were not texturized; their solubility values ranged from 71.8% to 92.6%. A wide possibility exists for creating new foods with texturized dairy proteins due to the extensive range of states achievable. Dairy proteins can be used to boost the protein content in puffed snacks made from corn meal, but unmodified, they bind water and form doughy pastes with starch. To minimize the water binding property of dairy proteins, WPI, or WPC, or NDM were modified by extrusion processing. Extrusion temperature conditions were adjusted to 50, 75, or 100 degrees C, sufficient to change the structure of the dairy proteins, but not destroy them. Extrusion modified the structures of these dairy proteins for ease of use in starchy foods to boost nutrient levels. Dairy proteins can be used to boost the protein content in puffed snacks made from corn meal, but unmodified, they bind water and form doughy pastes with starch. To minimize the water binding property of dairy proteins, whey protein isolate, whey protein concentrate, or nonfat dried milk were modified by extrusion processing. Extrusion temperature conditions were adjusted to 50, 75, or 100 degrees C, sufficient to change the structure of the dairy proteins, but not destroy them. Extrusion modified the structures of these dairy proteins for ease of use in starchy foods to boost nutrient levels.

The basic leucine zipper domain of c-Jun functions in transcriptional activation through interaction with the N terminus of human TATA-binding protein-associated factor-1 (human TAF(II)250).

PubMed

Lively, Tricia N; Nguyen, Tuan N; Galasinski, Shelly K; Goodrich, James A

2004-06-18

We previously reported that c-Jun binds directly to the N-terminal 163 amino acids of Homo sapiens TATA-binding protein-associated factor-1 (hsTAF1), causing a derepression of transcription factor IID (TFIID)-driven transcription (Lively, T. N., Ferguson, H. A., Galasinski, S. K., Seto, A. G., and Goodrich, J. A. (2001) J. Biol. Chem. 276, 25582-25588). This region of hsTAF1 binds TATA-binding protein to repress TFIID DNA binding and transcription. Here we show that the basic leucine zipper domain of c-Jun, which allows for DNA binding and homodimerization, is necessary and sufficient for interaction with hsTAF1. Interestingly, the isolated basic leucine zipper domain of c-Jun was able to derepress TFIID-directed basal transcription in vitro. Moreover, when the N-terminal region of hsTAF1 was added to in vitro transcription reactions and overexpressed in cells, it blocked c-Jun activation. c-Fos, another basic leucine zipper protein, did not interact with hsTAF1, but c-Fos/c-Jun heterodimers did bind the N terminus of hsTAF1. Our studies show that, in addition to dimerization and DNA binding, the well characterized basic leucine zipper domain of c-Jun functions in transcriptional activation by binding to the N terminus of hsTAF1 to derepress transcription.

A novel astaxanthin-binding photooxidative stress-inducible aqueous carotenoprotein from a eukaryotic microalga isolated from asphalt in midsummer.

PubMed

Kawasaki, Shinji; Mizuguchi, Keisuke; Sato, Masaru; Kono, Tetsuya; Shimizu, Hirofumi

2013-07-01

Water-soluble orange carotenoid proteins (OCPs) that bind 3'-hydroxyechinenone are found in cyanobacteria, and are thought to play a key role in photoprotection. The distribution of OCPs in eukaryotes remains largely unknown. In this study, we identified a novel OCP that predominantly binds astaxanthin from a eukaryotic microalga, strain Ki-4, isolated from a dry surface of heated asphalt in midsummer. A purified astaxanthin-binding OCP, named AstaP, shows high solubility in water with an absorption peak at 484 nm, and possesses a heat-stable activity that quenches singlet oxygen. The deduced amino acid sequence of AstaP comprises an N-terminal hydrophobic signal peptide, fasciclin domains found in secreted and cell surface proteins, and N-linked glycosylation sites, the first example of a carotenoprotein among fasciclin family proteins. AstaP homologs of unknown function are distributed mainly in organisms from the hydrosphere, such as marine bacteria, cyanobacteria, sea anemone and eukaryotic microalgae; however, AstaP exhibits a unique extraordinarily high isoelectric point (pI) value among homologs. The gene encoding AstaP, as well as the AstaP peptide, is expressed abundantly under conditions of dehydration and salt stress in conjunction with high light exposure. As a unique aqueous carotenoprotein, AstaP will provide a novel function of OCPs in protection against extreme photooxidative stresses.

The simulation approach to lipid-protein interactions.

PubMed

Paramo, Teresa; Garzón, Diana; Holdbrook, Daniel A; Khalid, Syma; Bond, Peter J

2013-01-01

The interactions between lipids and proteins are crucial for a range of biological processes, from the folding and stability of membrane proteins to signaling and metabolism facilitated by lipid-binding proteins. However, high-resolution structural details concerning functional lipid/protein interactions are scarce due to barriers in both experimental isolation of native lipid-bound complexes and subsequent biophysical characterization. The molecular dynamics (MD) simulation approach provides a means to complement available structural data, yielding dynamic, structural, and thermodynamic data for a protein embedded within a physiologically realistic, modelled lipid environment. In this chapter, we provide a guide to current methods for setting up and running simulations of membrane proteins and soluble, lipid-binding proteins, using standard atomistically detailed representations, as well as simplified, coarse-grained models. In addition, we outline recent studies that illustrate the power of the simulation approach in the context of biologically relevant lipid/protein interactions.

Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

PubMed

Nilsson, O Rickard; Lannergård, Jonas; Morgan, B Paul; Lindahl, Gunnar; Gustafsson, Mattias C U

2013-01-01

Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

PubMed Central

Robinson, Lois; Panayiotakis, Alexandra; Papas, Takis S.; Kola, Ismail; Seth, Arun

1997-01-01

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene. PMID:9207063

Cytoplasmic and nuclear localizations are important for the hypersensitive response conferred by maize autoactive Rp1-D21 protein

USDA-ARS?s Scientific Manuscript database

Disease resistance (R-) genes have been isolated from many plant species. Most encode nucleotide binding leucine-rich-repeat (NLR) proteins that trigger a rapid localized programmed cell death termed the hypersensitive response (HR) upon pathogen recognition. Despite their structural similarities, d...

Strains of Actinomyces naeslundii and Actinomyces viscosus Exhibit Structurally Variant Fimbrial Subunit Proteins and Bind to Different Peptide Motifs in Salivary Proteins

PubMed Central

Li, Tong; Johansson, Ingegerd; Hay, Donald I.; Strömberg, Nicklas

1999-01-01

Oral strains of Actinomyces spp. express type 1 fimbriae, which are composed of major FimP subunits, and bind preferentially to salivary acidic proline-rich proteins (APRPs) or to statherin. We have mapped genetic differences in the fimP subunit genes and the peptide recognition motifs within the host proteins associated with these differential binding specificities. The fimP genes were amplified by PCR from Actinomyces viscosus ATCC 19246, with preferential binding to statherin, and from Actinomyces naeslundii LY7, P-1-K, and B-1-K, with preferential binding to APRPs. The fimP gene from the statherin-binding strain 19246 is novel and has about 80% nucleotide and amino acid sequence identity to the highly conserved fimP genes of the APRP-binding strains (about 98 to 99% sequence identity). The novel FimP protein contains an amino-terminal signal peptide, randomly distributed single-amino-acid substitutions, and structurally different segments and ends with a cell wall-anchoring and a membrane-spanning region. When agarose beads with CNBr-linked host determinant-specific decapeptides were used, A. viscosus 19246 bound to the Thr42Phe43 terminus of statherin and A. naeslundii LY7 bound to the Pro149Gln150 termini of APRPs. Furthermore, while the APRP-binding A. naeslundii strains originate from the human mouth, A. viscosus strains isolated from the oral cavity of rat and hamster hosts showed preferential binding to statherin and contained the novel fimP gene. Thus, A. viscosus and A. naeslundii display structurally variant fimP genes whose protein products are likely to interact with different peptide motifs and to determine animal host tropism. PMID:10225854

Tumour-promoting phorbol esters increase basal and inhibit insulin-stimulated lipogenesis in rat adipocytes without decreasing insulin binding.

PubMed Central

van de Werve, G; Proietto, J; Jeanrenaud, B

1985-01-01

In isolated rat adipocytes, tumour-promoting phorbol esters caused (1) dose-dependent stimulation of lipogenesis in the absence of insulin and (2) inhibition of the lipogenic effect of submaximal concentrations of insulin, but without affecting insulin binding. The possible involvement of protein kinase C in insulin action is discussed. PMID:3883992

Dscam1 web server: online prediction of Dscam1 self- and hetero-affinity.

PubMed

Marini, Simone; Nazzicari, Nelson; Biscarini, Filippo; Wang, Guang-Zhong

2017-06-15

Formation of homodimers by identical Dscam1 protein isomers on cell surface is the key factor for the self-avoidance of growing neurites. Dscam1 immense diversity has a critical role in the formation of arthropod neuronal circuit, showing unique evolutionary properties when compared to other cell surface proteins. Experimental measures are available for 89 self-binding and 1722 hetero-binding protein samples, out of more than 19 thousands (self-binding) and 350 millions (hetero-binding) possible isomer combinations. We developed Dscam1 Web Server to quickly predict Dscam1 self- and hetero- binding affinity for batches of Dscam1 isomers. The server can help the study of Dscam1 affinity and help researchers navigate through the tens of millions of possible isomer combinations to isolate the strong-binding ones. Dscam1 Web Server is freely available at: http://bioinformatics.tecnoparco.org/Dscam1-webserver . Web server code is available at https://gitlab.com/ne1s0n/Dscam1-binding . simone.marini@unipv.it or guangzhong.wang@picb.ac.cn. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

CHMP6 and VPS4A mediate recycling of Ras to the plasma membrane to promote growth factor signaling

PubMed Central

Zheng, Ze-Yi; Cheng, Chiang-Min; Fu, Xin-Rong; Chen, Liuh-Yow; Xu, Lizhong; Terrillon, Sonia; Wong, Stephen T.; Bar-Sagi, Dafna; Songyang, Zhou; Chang, Eric C.

2011-01-01

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III, were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding-loop and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound N-Ras, but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked EGFR recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling. PMID:22231449

Landscape phages and their fusion proteins targeted to breast cancer cells

PubMed Central

Fagbohun, Olusegun A.; Bedi, Deepa; Grabchenko, Natalia I.; Deinnocentes, Patricia A.; Bird, Richard C.; Petrenko, Valery A.

2012-01-01

Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific ‘fusion pVIII proteins’ (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage–cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1. PMID:22490956

Potential of the Lectin/Inhibitor Isolated from Crataeva tapia Bark (CrataBL) for Controlling Callosobruchus maculatus Larva Development.

PubMed

Nunes, Natalia N S; Ferreira, Rodrigo S; Silva-Lucca, Rosemeire A; de Sá, Leonardo F R; de Oliveira, Antônia Elenir A; Correia, Maria Tereza dos S; Paiva, Patrícia Maria G; Wlodawer, Alexander; Oliva, Maria Luiza V

2015-12-09

Callosobruchus maculatus is an important predator of cowpeas. Due to infestation during storage, this insect affects the quality of seed and crop yield. This study aimed to investigate the effects of CrataBL, a multifunction protein isolated from Crataeva tapia bark, on C. maculatus larva development. The protein, which is stable even in extreme pH conditions, showed toxic activity, reducing the larval mass 45 and 70% at concentrations of 0.25 and 1.0% (w/w), respectively. Acting as an inhibitor, CrataBL decreased by 39% the activity of cysteine proteinases from larval gut. Conversely, the activity of serine proteinases was increased about 8-fold. The toxic properties of CrataBL may also be attributed to its capacity of binding to glycoproteins or glycosaminoglycans. Such binding interferes with larval metabolism, because CrataBL-FITC was found in the fat body, Malpighian tubules, and feces of larvae. These results demonstrate the potential of this protein for controlling larva development.

Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries.

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

Kay, B. K.; Castagnoli, L.; Biosciences Division

This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

Structure and Function of the Macrolide Biosensor Protein, MphR(A), with and without Erythromycin

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

Zheng, Jianting; Sagar, Vatsala; Smolinsky, Adam

2009-09-02

The regulatory protein MphR(A) has recently seen extensive use in synthetic biological applications, such as metabolite sensing and exogenous control of gene expression. This protein negatively regulates the expression of a macrolide 2{prime}-phosphotransferase I resistance gene (mphA) via binding to a 35-bp DNA operator upstream of the start codon and is de-repressed by the presence of erythromycin. Here, we present the refined crystal structure of the MphR(A) protein free of erythromycin and that of the MphR(A) protein with bound erythromycin at 2.00- and 1.76-{angstrom} resolutions, respectively. We also studied the DNA binding properties of the protein and identified mutants ofmore » MphR(A) that are defective in gene repression and ligand binding in a cell-based reporter assay. The combination of these two structures illustrates the molecular basis of erythromycin-induced gene expression and provides a framework for additional applied uses of this protein in the isolation and engineered biosynthesis of polyketide natural products.« less

Modeling disordered protein interactions from biophysical principles

PubMed Central

Christoffer, Charles; Terashi, Genki

2017-01-01

Disordered protein-protein interactions (PPIs), those involving a folded protein and an intrinsically disordered protein (IDP), are prevalent in the cell, including important signaling and regulatory pathways. IDPs do not adopt a single dominant structure in isolation but often become ordered upon binding. To aid understanding of the molecular mechanisms of disordered PPIs, it is crucial to obtain the tertiary structure of the PPIs. However, experimental methods have difficulty in solving disordered PPIs and existing protein-protein and protein-peptide docking methods are not able to model them. Here we present a novel computational method, IDP-LZerD, which models the conformation of a disordered PPI by considering the biophysical binding mechanism of an IDP to a structured protein, whereby a local segment of the IDP initiates the interaction and subsequently the remaining IDP regions explore and coalesce around the initial binding site. On a dataset of 22 disordered PPIs with IDPs up to 69 amino acids, successful predictions were made for 21 bound and 18 unbound receptors. The successful modeling provides additional support for biophysical principles. Moreover, the new technique significantly expands the capability of protein structure modeling and provides crucial insights into the molecular mechanisms of disordered PPIs. PMID:28394890

Fluctuations in Mass-Action Equilibrium of Protein Binding Networks

NASA Astrophysics Data System (ADS)

Yan, Koon-Kiu; Walker, Dylan; Maslov, Sergei

2008-12-01

We consider two types of fluctuations in the mass-action equilibrium in protein binding networks. The first type is driven by slow changes in total concentrations of interacting proteins. The second type (spontaneous) is caused by quickly decaying thermodynamic deviations away from equilibrium. We investigate the effects of network connectivity on fluctuations by comparing them to scenarios in which the interacting pair is isolated from the network and analytically derives bounds on fluctuations. Collective effects are shown to sometimes lead to large amplification of spontaneous fluctuations. The strength of both types of fluctuations is positively correlated with the complex connectivity and negatively correlated with complex concentration. Our general findings are illustrated using a curated network of protein interactions and multiprotein complexes in baker’s yeast, with empirical protein concentrations.

Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor.

PubMed Central

Ogunnariwo, J A; Schryvers, A B

1990-01-01

Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure. Images PMID:2365453

Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears.

PubMed

Kautto, Liisa; Nguyen-Khuong, Terry; Everest-Dass, Arun; Leong, Andrea; Zhao, Zhenjun; Willcox, Mark D P; Packer, Nicolle H; Peterson, Robyn

2016-04-01

The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached N-glycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain; 2) pre-incubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more binding inhibition than by the same amount of intact human lactoferrin or by the plant-derived N-glycans released from the rice recombinant lactoferrin; 3) pre-incubation of the bacteria with N-linked glycans released from human tear proteins inhibiting the adhesion of the ocular P. aeruginosa strains to immobilised tear proteins; 4) inhibition by the N-glycans from lactoferrin of the ability of an ocular strain of P. aeruginosa to invade corneal epithelial cells; 5) removal of terminal sialic acid and fucose moieties from the tear glycoproteins with α2-3,6,8 neuraminidase (sialidase) and α1-2,3,4 fucosidase resulting in a reduction in binding of the UTI P. aeruginosa isolate, but not the adhesion of the ocular cytotoxic (6206) or invasive (6294) isolates. Glycosidase activity was validated by mass spectrometry. In all cases, the magnitude of inhibition of bacterial adhesion by the N-glycans was consistently greater for the cytotoxic ocular strain than for the invasive ocular strain. Ocular P. aeruginosa isolates seems to exhibit different adhesion mechanism than previously known PAI and PAII lectin adhesion. The work may contribute towards the development of glycan-focused therapies to prevent P. aeruginosa infection of the eye. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

PubMed

Hubbell, H R; Rothblum, L I; Hsu, T C

1979-10-01

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.

Distribution of virulence factors and association with emm polymorphism or isolation site among beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis.

PubMed

Lo, Hsueh-Hsia; Cheng, Wei-Shan

2015-01-01

Distribution of virulence factors and association with emm polymorphism or isolation site among beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis. Streptococcus dysgalactiae subspecies equisimilis (SDSE), the dominant human pathogenic species among group G streptococci, is the causative agent of several invasive and non-invasive diseases worldwide. However, limited information is available about the distribution of virulence factors among SDSE isolates, or their association with emm types and the isolation sites. In this study, 246 beta-hemolytic group G SDSE isolates collected in central Taiwan between February 2007 and August 2011 were under investigation. Of these, 66 isolates were obtained from normally sterile sites and 180 from non-sterile sites. emm typing revealed 32 types, with the most prevalent one being stG10.0 (39.8%), followed by stG245.0 (15.4%), stG840.0 (12.2%), stG6.1 (7.7%), and stG652.0 (4.1%). The virulence genes lmb (encoding laminin-binding protein), gapC (glyceraldehyde 3-phosphate dehydrogenase), sagA (streptolysin S), and hylB (hyaluronidase) existed in all isolates. Also, 99.2% of the isolates possessed slo (streptolysin O) and scpA (C5a peptidase) genes. In addition, 72.8%, 14.6%, 9.4%, and 2.4% of the isolates possessed the genes ska (streptokinase), cbp (putative collagen-binding protein, SDEG_1781), fbp (putative fibronectin-binding protein, SDEG_0161), and sicG (streptococcal inhibitor of complement), respectively. The only superantigen gene detected was spegg (streptococcus pyrogenic exotoxin G(dys) ), which was possessed by 74.4% of the isolates; these isolates correlated with non-sterile sites. Positive correlations were observed between the following emm types and virulence genes: stG10.0 and stG840.0 with spegg, stG6.1 and stG652.0 with ska, and stG840.0 with cbp. On the other hand, negative correlations were observed between the following: stG245.0, stG6.1, and stG652.0 types with spegg, stG10.0 with ska, and stG10.0, stG245.0, and stG6.1 types with cbp. The prevalence of emm types of SDSE in central Taiwan was investigated for the first time. Moreover, the distribution of virulence factors among beta-hemolytic group G SDSE isolates, as well as their association with emm types or isolation sites were also examined. © 2014 APMIS. Published by John Wiley & Sons Ltd.

MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1.

PubMed

Lim, Raelene; Winteringham, Louise N; Williams, James H; McCulloch, Ross K; Ingley, Evan; Tiao, Jim Y-H; Lalonde, Jean-Philippe; Tsai, Schickwann; Tilbrook, Peta A; Sun, Yi; Wu, Xiaohua; Morris, Stephan W; Klinken, S Peter

2002-10-25

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

TALE-PvuII fusion proteins--novel tools for gene targeting.

PubMed

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

PubMed

Schilke, Brenda A; Ciesielski, Szymon J; Ziegelhoffer, Thomas; Kamiya, Erina; Tonelli, Marco; Lee, Woonghee; Cornilescu, Gabriel; Hines, Justin K; Markley, John L; Craig, Elizabeth A

2017-10-01

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

Isolation and Characterization of Adhesive Secretion from Cuvierian Tubules of Sea Cucumber Holothuria forskåli (Echinodermata: Holothuroidea)

PubMed Central

Baranowska, Malgorzata; Schloßmacher, Ute; McKenzie, J. Douglas; Müller, Werner E. G.; Schröder, Heinz C.

2011-01-01

The sea cucumber Holothuria forskåli possesses a specialized system called Cuvierian tubules. During mechanical stimulation white filaments (tubules) are expelled and become sticky upon contact with any object. We isolated a protein with adhesive properties from protein extracts of Cuvierian tubules from H. forskåli. This protein was identified by antibodies against recombinant precollagen D which is located in the byssal threads of the mussel Mytilus galloprovincialis. To find out the optimal procedure for extraction and purification, the identified protein was isolated by several methods, including electroelution, binding to glass beads, immunoprecipitation, and gel filtration. Antibodies raised against the isolated protein were used for localization of the adhesive protein in Cuvierian tubules. Immunostaining and immunogold electron microscopical studies revealed the strongest immunoreactivity in the mesothelium; this tissue layer is involved in adhesion. Adhesion of Cuvierian tubule extracts was measured on the surface of various materials. The extracted protein showed the strongest adhesion to Teflon surface. Increased adhesion was observed in the presence of potassium and EDTA, while cadmium caused a decrease in adhesion. Addition of antibodies and trypsin abolished the adhesive properties of the extract. PMID:22013488

Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1).

PubMed

McDermott, M J; Weber, E; Hunter, S; Stedman, K E; Best, E; Frank, G R; Wang, R; Escudero, J; Kuner, J; McCall, C

2000-05-01

An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.

The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

PubMed

Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

2007-01-01

The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA.

PubMed

Lai, Ruenn Chai; Tan, Soon Sim; Yeo, Ronne Wee Yeh; Choo, Andre Boon Hwa; Reiner, Agnes T; Su, Yan; Shen, Yang; Fu, Zhiyan; Alexander, Lezhava; Sze, Siu Kwan; Lim, Sai Kiang

2016-01-01

Mesenchymal stem cell (MSC), a widely used adult stem cell candidate for regenerative medicine, has been shown to exert some of its therapeutic effects through the secretion of extracellular vesicles (EVs). These homogenously sized EVs of 100-150 ηm exhibited many exosome-like biophysical and biochemical properties and carry both proteins and RNAs. Recently, exosome-associated proteins in this MSC EV preparation were found to segregate primarily to those EVs that bind cholera toxin B chain (CTB), a GM1 ganglioside-specific ligand, and pulse-chase experiments demonstrated that these EVs have endosomal origin and carried many of the exosome-associated markers. Here, we report that only a fraction of the MSC EV proteome was found in CTB-bound EVs. Using Annexin V (AV) and Shiga toxin B subunit (ST) with affinities for phosphatidylserine and globotriaosylceramide, respectively, AV- and a ST-binding EV were identified. CTB-, AV- and ST-binding EVs all carried actin. However, the AV-binding EVs carried low or undetectable levels of the exosome-associated proteins. Only the ST-binding EVs carried RNA and EDA-containing fibronectin. Proteins in AV-binding EVs were also different from those released by apoptotic MSCs. CTB- and AV-binding activities were localized to the plasma membrane and cytoplasm of MSCs, while ST-binding activity was localized to the nucleus. Together, this study demonstrates that cells secrete many types of EVs. Specifically, MSCs secrete at least 3 types. They can be differentially isolated based on their affinities for membrane lipid-binding ligands. As the subcellular sites of the binding activities of these ligands and cargo load are different for each EV type, they are likely to have a different biogenesis pathway and possibly different functions.

Isolation and cDNA cloning of a novel red colour-related pigment-binding protein derived from the shell of the shrimp, Litopenaeus vannamei.

PubMed

Pan, Chuang; Ishizaki, Shoichiro; Nagashima, Yuji; Gao, Jialong; Watabe, Shugo

2018-02-15

Pigment-binding proteins play important roles in crustacean shell colour change. In this study, a red colour-related pigment-binding protein, designated LvPBP75, was purified from the shell of Litopenaeus vannamei. HPLC and PAGE analysis showed that LvPBP75 was a homogeneous monomer with molecular mass of 75kDa. Peptide mass fingerprint analysis revealed that LvPBP75 belonged to hemocyanin, and the released pigment from heated LvPBP75 showed a λ max at 481nm in acetone. The significant red-colour change temperatures were detected at 30 and 80°C, respectively. Based on the determined amino acid fragments, a full-length cDNA of LvPBP75 was cloned and sequenced. The ORF encodes a protein of 662 amino acids having 80% identity with penaeidae hemocyanin. These results strongly suggest a novel function of hemocyanin, namely binding with pigment, and its involvement in L. vannamei shell colour change. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cysteine Protease-Binding Protein Family 6 Mediates the Trafficking of Amylases to Phagosomes in the Enteric Protozoan Entamoeba histolytica

PubMed Central

Furukawa, Atsushi; Nakada-Tsukui, Kumiko

2013-01-01

Phagocytosis plays a pivotal role in nutrient acquisition and evasion from the host defense systems in Entamoeba histolytica, the intestinal protozoan parasite that causes amoebiasis. We previously reported that E. histolytica possesses a unique class of a hydrolase receptor family, designated the cysteine protease-binding protein family (CPBF), that is involved in trafficking of hydrolases to lysosomes and phagosomes, and we have also reported that CPBF1 and CPBF8 bind to cysteine proteases or β-hexosaminidase α-subunit and lysozymes, respectively. In this study, we showed by immunoprecipitation that CPBF6, one of the most highly expressed CPBF proteins, specifically binds to α-amylase and γ-amylase. We also found that CPBF6 is localized in lysosomes, based on immunofluorescence imaging. Immunoblot and proteome analyses of the isolated phagosomes showed that CPBF6 mediates transport of amylases to phagosomes. We also demonstrated that the carboxyl-terminal cytosolic region of CPBF6 is engaged in the regulation of the trafficking of CPBF6 to phagosomes. Our proteome analysis of phagosomes also revealed new potential phagosomal proteins. PMID:23509141

A cDNA from a mouse pancreatic beta cell encoding a putative transcription factor of the insulin gene.

PubMed Central

Walker, M D; Park, C W; Rosen, A; Aronheim, A

1990-01-01

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on multiple DNA sequence elements located in the 5' flanking region of the gene. Of particular importance in the rat insulin I gene are two closely similar 9 bp sequences (IEB1 and IEB2): mutation of either of these leads to 5-10 fold reduction in transcriptional activity. We have screened an expression cDNA library derived from mouse pancreatic endocrine beta cells with a radioactive DNA probe containing multiple copies of the IEB1 sequence. A cDNA clone (A1) isolated by this procedure encodes a protein which shows efficient binding to the IEB1 probe, but much weaker binding to either an unrelated DNA probe or to a probe bearing a single base pair insertion within the recognition sequence. DNA sequence analysis indicates a protein belonging to the helix-loop-helix family of DNA-binding proteins. The ability of the protein encoded by clone A1 to recognize a number of wild type and mutant DNA sequences correlates closely with the ability of each sequence element to support transcription in vivo in the context of the insulin 5' flanking DNA. We conclude that the isolated cDNA may encode a transcription factor that participates in control of insulin gene expression. Images PMID:2181401

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

PubMed

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

1995-03-03

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

Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

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

Deshpande, Chandrika N.; Harrop, Stephen J.; Boucher, Yan

2012-02-15

The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. We report the 1.8 {angstrom} crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, suchmore » as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.« less

Protein-Protein Interactions in the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL from Azotobacter vinelandii

PubMed Central

Money, Tracy; Barrett, Jason; Dixon, Ray; Austin, Sara

2001-01-01

The enhancer binding protein NIFA and the sensor protein NIFL from Azotobacter vinelandii comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. Using limited proteolysis of the purified proteins, we show here that changes in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred when the complex was formed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protected by NIFL. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are not detectable by cochromatography. PMID:11157949

Antioxidant and Antiplasmodial Activities of Bergenin and 11-O-Galloylbergenin Isolated from Mallotus philippensis

PubMed Central

Khan, Hamayun; Amin, Hazrat; Ullah, Asad; Saba, Sumbal; Rafique, Jamal; Khan, Khalid; Ahmad, Nasir; Badshah, Syed Lal

2016-01-01

Two important biologically active compounds were isolated from Mallotus philippensis. The isolated compounds were characterized using spectroanalytical techniques and found to be bergenin (1) and 11-O-galloylbergenin (2). The in vitro antioxidant and antiplasmodial activities of the isolated compounds were determined. For the antioxidant potential, three standard analytical protocols, namely, DPPH radical scavenging activity (RSA), reducing power assay (RPA), and total antioxidant capacity (TAC) assay, were adopted. The results showed that compound 2 was found to be more potent antioxidant as compared to 1. Fascinatingly, compound 2 displayed better EC50 results as compared to α-tocopherol while being comparable with ascorbic acid. The antiplasmodial assay data showed that both the compound exhibited good activity against chloroquine sensitive strain of Plasmodium falciparum (D10) and IC50 values were found to be less than 8 μM. The in silico molecular docking analyses were also performed for the determination of binding affinity of the isolated compounds using P. falciparum proteins PfLDH and Pfg27. The results showed that compound 2 has high docking score and binding affinity to both protein receptors as compared to compound 1. The demonstrated biological potentials declared that compound 2 could be the better natural antioxidant and antiplasmodial candidate. PMID:26998192

DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

PubMed

Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

2017-09-01

Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

New Mutations of Penicillin-Binding Proteins in Streptococcus agalactiae Isolates from Cattle with Decreased Susceptibility to Penicillin.

PubMed

Hu, Yun; Kan, Yunchao; Zhang, Zhengtian; Lu, Zhanning; Li, Yanqiu; Leng, Chaoliang; Ji, Jun; Song, Shiyang; Shi, Hongfei

2018-02-23

Streptococcus agalactiae is a causal agent of bovine mastitis and is treated by β-lactam antibiotics (BLAs). Compared to penicillin-resistant S. agalactiae from humans, resistant strains in bovine are rarely reported. In this study, we aimed to investigate BLA resistance and mutations in penicillin-binding proteins (PBPs) of S. agalactiae in central and northeast China. The minimum inhibitory concentrations (MICs) of 129 penicillin-resistant S. agalactiae isolates from cows with mastitis were determined, and the related PBP genes were detected and sequenced. All strains were unsusceptible to penicillin G and mostly resistant to ampicillin, cefalexin, and ceftiofur sodium. One hundred twenty-nine strains were divided into 4 clonal groups and 8 sequence types by multilocus sequence typing analysis. We found a set of new substitutions in PBP1B, PBP2B, and PBP2X from most strains isolated from three provinces. The strains with high PBP mutations showed a broader unsusceptible spectrum and higher MICs than those with few or single mutation. Our research indicates unpredicted mutations in the PBP genes of S. agalactiae isolated from cows with mastitis treated by BLAs. This screening is the first of S. agalactiae from cattle.

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Decoding the patterns of ubiquitin recognition by ubiquitin-associated domains from free energy simulations.

PubMed

Bouvier, Benjamin

2014-01-07

Ubiquitin is a highly conserved, highly represented protein acting as a regulating signal in numerous cellular processes. It leverages a single hydrophobic binding patch to recognize and bind a large variety of protein domains with remarkable specificity, but can also self-assemble into chains of poly-diubiquitin units in which these interfaces are sequestered, profoundly altering the individual monomers' recognition characteristics. Despite numerous studies, the origins of this varied specificity and the competition between substrates for the binding of the ubiquitin interface patch remain under heated debate. This study uses enhanced sampling all-atom molecular dynamics to simulate the unbinding of complexes of mono- or K48-linked diubiquitin bound to several ubiquitin-associated domains, providing insights into the mechanism and free energetics of ubiquitin recognition and binding. The implications for the subtle tradeoff between the stability of the polyubiquitin signal and its easy recognition by target protein assemblies are discussed, as is the enhanced affinity of the latter for long polyubiquitin chains compared to isolated mono- or diubiquitin.

Total external reflection X-ray fluorescence analysis of protein-metal ion interactions in biological systems

NASA Astrophysics Data System (ADS)

Novikova, N. N.; Kovalchuk, M. V.; Yur'eva, E. A.; Konovalov, O. V.; Rogachev, A. V.; Stepina, N. D.; Sukhorukov, V. S.; Tsaregorodtsev, A. D.; Chukhrai, E. S.; Yakunin, S. N.

2012-09-01

This paper presents the results of an investigation into hemoglobin-based protein films that were formed on a liquid surface. X-ray standing wave measurements were performed at the ID 10 beamline of the European Synchrotron Radiation Facility (ESRF) and at the Langmuir station of the Kurchatov Synchrotron Radiation Source. It was found that the ability of the protein to bind metal ions is substantially increased due to the conformational rearrangements of protein macromolecules caused by various damaging effects. The elemental composition of protein preparations, which were isolated from children and adults with chronic metabolic diseases accompanied by endogenous intoxication, was analyzed. The results of the investigations offer evidence that an increase in the ligand-binding properties of the protein molecules, which was observed in model experiments using protein films, is a common trait and corresponds to in vivo processes accompanying metabolic disturbances in the body.

Selective isolation of hydrophobin SC3 by solid-phase extraction with polytetrafluoroethylene microparticles and subsequent mass spectrometric analysis.

PubMed

Kupčík, Rudolf; Zelená, Miroslava; Řehulka, Pavel; Bílková, Zuzana; Česlová, Lenka

2016-02-01

Hydrophobins are small proteins that play a role in a number of processes during the filamentous fungi growth and development. These proteins are characterized by the self-assembly of their molecules into an amphipathic membrane at hydrophilic-hydrophobic interfaces. Isolation and purification of hydrophobins generally present a challenge in their analysis. Hydrophobin SC3 from Schizophyllum commune was selected as a representative of class I hydrophobins in this work. A novel procedure for selective and effective isolation of hydrophobin SC3 based on solid-phase extraction with polytetrafluoroethylene microparticles loaded in a small self-made microcolumn is reported. The tailored binding of hydrophobins to polytetrafluoroethylene followed by harsh elution conditions resulted in a highly specific isolation of hydrophobin SC3 from the model mixture of ten proteins. The presented isolation protocol can have a positive impact on the analysis and utilization of these proteins including all class I hydrophobins. Hydrophobin SC3 was further subjected to reduction of its highly stable disulfide bonds and to chymotryptic digestion followed by mass spectrometric analysis. The isolation and digestion protocols presented in this work make the analysis of these highly hydrophobic and compact proteins possible. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Yrb4p, a yeast ran-GTP-binding protein involved in import of ribosomal protein L25 into the nucleus.

PubMed Central

Schlenstedt, G; Smirnova, E; Deane, R; Solsbacher, J; Kutay, U; Görlich, D; Ponstingl, H; Bischoff, F R

1997-01-01

Gsp1p, the essential yeast Ran homologue, is a key regulator of transport across the nuclear pore complex (NPC). We report the identification of Yrb4p, a novel Gsp1p binding protein. The 123 kDa protein was isolated from Saccharomyces cerevisiae cells and found to be related to importin-beta, the mediator of nuclear localization signal (NLS)-dependent import into the nucleus, and to Pse1p. Like importin-beta, Yrb4p and Pse1p specifically bind to Gsp1p-GTP, protecting it from GTP hydrolysis and nucleotide exchange. The GTPase block of Gsp1p complexed to Yrb4p or Pse1p is released by Yrb1p, which contains a Gsp1p binding domain distinct from that of Yrb4p. This might reflect an in vivo function for Yrb1p. Cells disrupted for YRB4 are defective in nuclear import of ribosomal protein L25, but show no defect in the import of proteins containing classical NLSs. Expression of a Yrb4p mutant deficient in Gsp1p-binding is dominant-lethal and blocks bidirectional traffic across the NPC in wild-type cells. L25 binds to Yrb4p and Pse1p and is released by Gsp1p-GTP. Consistent with its putative role as an import receptor for L25-like proteins, Yrb4p localizes to the cytoplasm, the nucleoplasm and the NPC. PMID:9321403

A root bond between ice and antifreeze protein.

PubMed

Hawes, Timothy C

2016-10-01

It has always been assumed that a three-dimensional protein structure is essential to antifreeze protein (AFP) ice interactions. Using a 9 kDa AFP isolated from the springtail, Gomphiocephalus hodgsoni, it was found that the bond between ice and protein is maintained independent of higher order protein structure. GomplyAFP9 remained bound to ice after denaturing by a range of agents (boiling, extreme pH, DTT, ethanol, urea). Thermal hysteresis was minimal (0.03-0.04 °C), but not lost. Crystal faceting and growth occurred normal to the c-axis, indicating the protein binds primarily to sites along the a-axis. These observations lend additional support to the hypothesis of irreversible binding. More significantly, they suggest that binding to ice and functional hysteresis may be achieved independently (i.e. are different operations). These results are consistent with the view that there is a root bond with ice and it is achieved via an amino acid derived interface that bonds to water molecules in aqueous solutions. Copyright © 2016 Elsevier Inc. All rights reserved.

Differences between culture & non-culture confirmed invasive meningococci with a focus on factor H-binding protein distribution.

PubMed

Clark, Stephen A; Lekshmi, Aiswarya; Lucidarme, Jay; Hao, Li; Tsao, How; Lee-Jones, Lisa; Jansen, Kathrin U; Newbold, Lynne S; Anderson, Annaliesa S; Borrow, Ray

2016-07-01

To compare the distribution of capsular groups and factor H-binding protein (fHBP) variants among meningococcal isolates and non-culture clinical specimens and to assess the representativeness of group B isolates amongst group B cases as a whole. A PCR sequencing assay was used to characterise fHBP from non-culture cases confirmed from January 2011 to December 2013. These were compared to genotypic data derived from whole genome analysis of isolates received during the same period. Group W and Y strains were more common among isolates than non-culture strains. The distribution of fHBP variants among group B non-culture cases generally reflected that seen in the corresponding isolates. Nonetheless, the non-culture subset contained a greater proportion of fHBP variant 15/B44, associated with the ST-269 cluster sublineage. Differences in capsular group and fHBP distribution among culture and non-culture cases may be indicative of variation in strain viability, diagnostic practice, disease severity and/or clinical presentation. Future analyses combining clinical case information with laboratory data may help to further explore these differences. Group B isolates provide a good representation of group B disease in E&W and, therefore, can reliably be used in fHBP strain coverage predictions of recently-licensed vaccines. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Nucleoplasmin-like domain of FKBP39 from Drosophila melanogaster forms a tetramer with partly disordered tentacle-like C-terminal segments

PubMed Central

Kozłowska, Małgorzata; Tarczewska, Aneta; Jakób, Michał; Bystranowska, Dominika; Taube, Michał; Kozak, Maciej; Czarnocki-Cieciura, Mariusz; Dziembowski, Andrzej; Orłowski, Marek; Tkocz, Katarzyna; Ożyhar, Andrzej

2017-01-01

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a β-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins. PMID:28074868

Binding of Thyrotropin-Releasing Hormone to Plasma Membranes of Bovine Anterior Pituitary Gland

PubMed Central

Labrie, Fernand; Barden, Nicholas; Poirier, Guy; De Lean, Andre

1972-01-01

An assay for the binding of [3H]thyrotropin-releasing hormone ([3H]TRH) is described. Plasma membranes isolated from bovine anterior pituitary gland bind about 600 femtomoles of this hormone per mg of protein, as compared to 15 femtomoles per mg of protein in the total adenohypophyseal homogenate (40-fold purification). The equilibrium constant of membrane receptor-[3H]TRH binding at 0°C is 4.3 × 107 L·M-1, or a half-maximal binding of this hormone at 23 nM. The binding is time-dependent; addition of unlabeled hormone induces dissociation of the receptor-[3H]TRH complex with a half-life of 14 min. The binding of TRH is not altered by 10 μM melanocyte-stimulating hormone-release inhibiting hormone, lysine-vasopressin, adrenocorticotropin, growth hormone, prolactin, luteinizing hormone, insulin, glucagon, L-thyroxine, or L-triiodothyronine. K+ and Mg++ increase formation of the receptor-TRH complex at optimal concentrations of 5-25 mM and 0.5-2.5 mM, respectively, with inhibition at higher concentrations. Ca++ inhibits binding of TRH at all concentrations tested. PMID:4621548

ZP Domain Proteins in the Abalone Egg Coat Include a Paralog of VERL under Positive Selection That Binds Lysin and 18-kDa Sperm Proteins

PubMed Central

Aagaard, Jan E.; Vacquier, Victor D.; MacCoss, Michael J.; Swanson, Willie J.

2010-01-01

Identifying fertilization molecules is key to our understanding of reproductive biology, yet only a few examples of interacting sperm and egg proteins are known. One of the best characterized comes from the invertebrate archeogastropod abalone (Haliotis spp.), where sperm lysin mediates passage through the protective egg vitelline envelope (VE) by binding to the VE protein vitelline envelope receptor for lysin (VERL). Rapid adaptive divergence of abalone lysin and VERL are an example of positive selection on interacting fertilization proteins contributing to reproductive isolation. Previously, we characterized a subset of the abalone VE proteins that share a structural feature, the zona pellucida (ZP) domain, which is common to VERL and the egg envelopes of vertebrates. Here, we use additional expressed sequence tag sequencing and shotgun proteomics to characterize this family of proteins in the abalone egg VE. We expand 3-fold the number of known ZP domain proteins present within the VE (now 30 in total) and identify a paralog of VERL (vitelline envelope zona pellucida domain protein [VEZP] 14) that contains a putative lysin-binding motif. We find that, like VERL, the divergence of VEZP14 among abalone species is driven by positive selection on the lysin-binding motif alone and that these paralogous egg VE proteins bind a similar set of sperm proteins including a rapidly evolving 18-kDa paralog of lysin, which may mediate sperm–egg fusion. This work identifies an egg coat paralog of VERL under positive selection and the candidate sperm proteins with which it may interact during abalone fertilization. PMID:19767347

Molecular Profiling of EPI-001: An Inhibitor of Androgen Receptor Signaling With a Disputed Mechanism of Action

DTIC Science & Technology

2016-09-01

analogues and to determine their protein - binding partners in prostate cancer cell lines. The anticipated outcome is the annotation of the activity...associated protein targets of EPI-001 that will be valuable in future drug development efforts. The progress achieved during this 1-year award include...cells to afford probe- protein adducts, (3) optimizing protocols for the isolation of probe- protein adducts over monomeric avidin resin and demonstrating

Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates

PubMed Central

O’Meally, Robert; Sonnenberg, Jason L.; Cole, Robert N.; Shewmaker, Frank P.

2015-01-01

Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (≥100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases. PMID:26317359

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries.

PubMed

Tullila, Antti; Nevanen, Tarja K

2017-05-31

Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

PubMed Central

Tullila, Antti; Nevanen, Tarja K.

2017-01-01

Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library. PMID:28561803

High-Affinity Interaction between the S-Layer Protein SbsC and the Secondary Cell Wall Polymer of Geobacillus stearothermophilus ATCC 12980 Determined by Surface Plasmon Resonance Technology▿ †

PubMed Central

Ferner-Ortner, Judith; Mader, Christoph; Ilk, Nicola; Sleytr, Uwe B.; Egelseer, Eva M.

2007-01-01

Surface plasmon resonance studies using C-terminal truncation forms of the S-layer protein SbsC (recombinant SbsC consisting of amino acids 31 to 270 [rSbsC31-270] and rSbsC31-443) and the secondary cell wall polymer (SCWP) isolated from Geobacillus stearothermophilus ATCC 12980 confirmed the exclusive responsibility of the N-terminal region comprising amino acids 31 to 270 for SCWP binding. Quantitative analyses indicated binding behavior demonstrating low, medium, and high affinities. PMID:17644609

Isolation, purification, crystallization and preliminary X-ray studies of two 30 kDa proteins from silkworm haemolymph.

PubMed

Pietrzyk, Agnieszka J; Bujacz, Anna; Łochyńska, Małgorzata; Jaskólski, Mariusz; Bujacz, Grzegorz

2011-03-01

Juvenile hormone-binding protein (JHBP) and the low-molecular-mass lipoprotein PBMHP-12 belong to a group of 30 kDa proteins that comprise the major protein component of the haemolymph specific to the fifth-instar larvae stage of the mulberry silkworm Bombyx mori L. Proteins from this group are often essential for the development of the insect. In a project aimed at crystallographic characterization of B. mori JHBP (BmJHBP), it was copurified together with PBMHP-12. Eventually, the two proteins were isolated and crystallized separately. The BmJHBP crystals were orthorhombic (space group C222(1)) and the PBMHP-12 crystals were triclinic. The crystals diffracted X-rays to 2.9 Å (BmJHBP) and 1.3 Å (PBMHP-12) resolution.

On the molecular interaction between lactoferrin and the dye Red HE-3B. A novel approach for docking a charged and highly flexible molecule to protein surfaces

NASA Astrophysics Data System (ADS)

Grasselli, Mariano; Cascone, Osvaldo; Anspach, F. Birger; Delfino, Jose M.

2002-12-01

Lactoferrin (Lf) is a non-heme, iron binding protein present in many physiological fluids of vertebrates where its main role is the microbicidal activity. It has been isolated by different methods, including dye-affinity chromatography. Red HE-3B is one of the most common triazinic dyes applied in protein purification, but scant knowledge is available on structural details and on the energetics of its interaction with proteins. In this work we present a computational approach useful for identifying possible binding sites for Red HE-3B in apo and holo forms of Lfs from human and bovine source. A new geometrical description of Red HE-3B is introduced which greatly simplifies the conformational analysis. This approach proved to be of particular advantage for addressing conformational ensembles of highly flexible molecules. Predictions from this analysis were correlated with experimentally observed dye-binding sites, as mapped by protection from proteolysis in Red HE-3B/Lf complexes. This method could bear relevance for the screening of possible dye-binding sites in proteins whose structure is known and as a potential tool for the design of engineered protein variants which could be purified by dye-affinity chromatography.

On the molecular interaction between lactoferrin and the dye Red HE-3b. A novel approach for docking a charged and highly flexible molecule to protein surfaces.

PubMed

Grasselli, Mariano; Cascone, Osvaldo; Birger Anspach, F; Delfino, Jose M

2002-12-01

Lactoferrin (Lf) is a non-heme, iron binding protein present in many physiological fluids of vertebrates where its main role is the microbicidal activity. It has been isolated by different methods, including dye-affinity chromatography. Red HE-3B is one of the most common triazinic dyes applied in protein purification, but scant knowledge is available on structural details and on the energetics of its interaction with proteins. In this work we present a computational approach useful for identifying possible binding sites for Red HE-3B in apo and holo forms of Lfs from human and bovine source. A new geometrical description of Red HE-3B is introduced which greatly simplifies the conformational analysis. This approach proved to be of particular advantage for addressing conformational ensembles of highly flexible molecules. Predictions from this analysis were correlated with experimentally observed dye-binding sites, as mapped by protection from proteolysis in Red HE-3B/Lf complexes. This method could bear relevance for the screening of possible dye-binding sites in proteins whose structure is known and as a potential tool for the design of engineered protein variants which could be purified by dye-affinity chromatography.

Isolation of serotype-specific antibodies against dengue virus non-structural protein 1 using phage display and application in a multiplexed serotyping assay.

PubMed

Lebani, Kebaneilwe; Jones, Martina L; Watterson, Daniel; Ranzoni, Andrea; Traves, Renee J; Young, Paul R; Mahler, Stephen M

2017-01-01

The multidimensional nature of dengue virus (DENV) infections, which can be caused by four distinct serotypes of the virus, complicates the sensitivity of assays designed for the diagnosis of infection. Different viral markers can be optimally detected at different stages of infection. Of particular clinical importance is the early identification of infection, which is pivotal for disease management and the development of blood screening assays. Non-structural protein 1 (NS1) is an early surrogate marker of infection and its detection in serum coincides with detectable viraemia. The aim of this work was to isolate and characterise serotype-specific monoclonal antibodies that bind to NS1 for each of the four DENV serotypes. This was achieved using phage display and a subtractive biopanning strategy to direct the antibody selection towards serotype-specific epitopes. This antibody isolation strategy has advantages over immunisation techniques where it is difficult to avoid antibody responses to cross-reactive, immunodominant epitopes. Serotype specificity to recombinant antigen for each of the antibodies was confirmed by Enzyme Linked Immunosorbent Assay (ELISA) and Surface Plasmon Resonance. Confirmation of binding to native DENV NS1 was achieved using ELISA and immunofluorescence assay on DENV infected Vero cells. No cross-reactivity with Zika or Kunjin viruses was observed. A previously isolated pan-reactive antibody that binds to an immunodominant epitope was able to pair with each of the serotype-specific antibodies in a sandwich ELISA, indicating that the serotype specific antibodies bind to epitopes which are all spatially distinct from the immunodominant epitope. These antibodies were suitable for use in a multiplexed assay for simultaneous detection and serotyping of DENV NS1 in human serum. This work demonstrates that phage display coupled with novel biopanning strategies is a valuable in vitro methodology for isolation of binders that can discern amongst antigens with high homology for diagnostic applicability.

Role of tyrosine hot-spot residues at the interface of colicin E9 and immunity protein 9: a comparative free energy simulation study.

PubMed

Luitz, Manuel P; Zacharias, Martin

2013-03-01

The endonuclease activity of the bacterial colicin 9 enzyme is controlled by the specific and high-affinity binding of immunity protein 9 (Im9). Molecular dynamics simulation studies in explicit solvent were used to investigate the free energy change associated with the mutation of two hot-spot interface residues [tyrosine (Tyr): Tyr54 and Tyr55] of Im9 to Ala. In addition, the effect of several other mutations (Leu33Ala, Leu52Ala, Val34Ala, Val37Ala, Ser48Ala, and Ile53Ala) with smaller influence on binding affinity was also studied. Good qualitative agreement of calculated free energy changes and experimental data on binding affinity of the mutations was observed. The simulation studies can help to elucidate the molecular details on how the mutations influence protein-protein binding affinity. The role of solvent and conformational flexibility of the partner proteins was studied by comparing the results in the presence or absence of solvent and with or without positional restraints. Restriction of the conformational mobility of protein partners resulted in significant changes of the calculated free energies but of similar magnitude for isolated Im9 and for the complex and therefore in only modest changes of binding free energy differences. Although the overall binding free energy change was similar for the two Tyr-Ala mutations, the physical origin appeared to be different with solvation changes contributing significantly to the Tyr55Ala mutation and to a loss of direct protein-protein interactions dominating the free energy change due to the Tyr54Ala mutation. Copyright © 2012 Wiley Periodicals, Inc.

A Gene Encoding a Hevein-Like Protein from Elderberry Fruits Is Homologous to PR-4 and Class V Chitinase Genes1

PubMed Central

Van Damme, Els J.M.; Charels, Diana; Roy, Soma; Tierens, Koenraad; Barre, Annick; Martins, José C.; Rougé, Pierre; Van Leuven, Fred; Does, Mirjam; Peumans, Willy J.

1999-01-01

We isolated SN-HLPf (Sambucus nigra hevein-like fruit protein), a hevein-like chitin-binding protein, from mature elderberry fruits. Cloning of the corresponding gene demonstrated that SN-HLPf is synthesized as a chimeric precursor consisting of an N-terminal chitin-binding domain corresponding to the mature elderberry protein and an unrelated C-terminal domain. Sequence comparisons indicated that the N-terminal domain of this precursor has high sequence similarity with the N-terminal domain of class I PR-4 (pathogenesis-related) proteins, whereas the C terminus is most closely related to that of class V chitinases. On the basis of these sequence homologies the gene encoding SN-HLPf can be considered a hybrid between a PR-4 and a class V chitinase gene. PMID:10198114

VAR2CSA domains expressed in Escherichia coli induce cross-reactive antibodies to native protein.

PubMed

Oleinikov, Andrew V; Francis, Susan E; Dorfman, Jeffrey R; Rossnagle, Eddie; Balcaitis, Stephanie; Getz, Tony; Avril, Marion; Gose, Severin; Smith, Joseph D; Fried, Michal; Duffy, Patrick E

2008-04-15

The variant surface antigen VAR2CSA is a pregnancy malaria vaccine candidate, but its size and polymorphism are obstacles to development. We expressed 3D7-type VAR2CSA domains in Escherichia coli as insoluble His-tagged proteins (Duffy binding-like [DBL] domains DBL1, DBL3, DBL4, and DBL5) that were denatured and refolded or as soluble glutathione S-transferase-tagged protein (DBL6). Anti-DBL5 antiserum cross-reacted with surface proteins of chondroitin sulfate A (CSA)-binding laboratory strains (3D7-CSA and FCR3-CSA) and a clinical pregnancy malaria isolate, whereas anti-DBL6 antiserum reacted only to 3D7 surface protein. This is the first report that E. coli-expressed VAR2CSA domains induce antibody to native VAR2CSA.

Evidence for Globally Shared, Cross-Reacting Polymorphic Epitopes in the Pregnancy-Associated Malaria Vaccine Candidate VAR2CSA▿

PubMed Central

Avril, Marion; Kulasekara, Bridget R.; Gose, Severin O.; Rowe, Chris; Dahlbäck, Madeleine; Duffy, Patrick E.; Fried, Michal; Salanti, Ali; Misher, Lynda; Narum, David L.; Smith, Joseph D.

2008-01-01

Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size (∼350 kDa) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three recombinant proteins (DBL1, DBL3, and DBL6) and four plasmid DNAs (DBL1, DBL3, DBL5, and DBL6) reacted with homologous FCR3-CSA IEs. By comparison, antibodies to the DBL4 domain were unable to react with native VAR2CSA protein unless it was first partially proteolyzed with trypsin or chymotrypsin. To investigate the antigenic relationship of geographically diverse CSA-binding isolates, rabbit immune sera were screened on four heterologous CSA-binding lines from different continental origins. Antibodies did not target conserved epitopes exposed in all VAR2CSA alleles; however, antisera to several DBL domains cross-reacted on parasite isolates that had polymorphic loops in common with the homologous immunogen. This study demonstrates that VAR2CSA contains common polymorphic epitopes that are shared between geographically diverse CSA-binding lines. PMID:18250177

Evidence for globally shared, cross-reacting polymorphic epitopes in the pregnancy-associated malaria vaccine candidate VAR2CSA.

PubMed

Avril, Marion; Kulasekara, Bridget R; Gose, Severin O; Rowe, Chris; Dahlbäck, Madeleine; Duffy, Patrick E; Fried, Michal; Salanti, Ali; Misher, Lynda; Narum, David L; Smith, Joseph D

2008-04-01

Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size ( approximately 350 kDa) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three recombinant proteins (DBL1, DBL3, and DBL6) and four plasmid DNAs (DBL1, DBL3, DBL5, and DBL6) reacted with homologous FCR3-CSA IEs. By comparison, antibodies to the DBL4 domain were unable to react with native VAR2CSA protein unless it was first partially proteolyzed with trypsin or chymotrypsin. To investigate the antigenic relationship of geographically diverse CSA-binding isolates, rabbit immune sera were screened on four heterologous CSA-binding lines from different continental origins. Antibodies did not target conserved epitopes exposed in all VAR2CSA alleles; however, antisera to several DBL domains cross-reacted on parasite isolates that had polymorphic loops in common with the homologous immunogen. This study demonstrates that VAR2CSA contains common polymorphic epitopes that are shared between geographically diverse CSA-binding lines.

HITS-CLIP yields genome-wide insights into brain alternative RNA processing

NASA Astrophysics Data System (ADS)

Licatalosi, Donny D.; Mele, Aldo; Fak, John J.; Ule, Jernej; Kayikci, Melis; Chi, Sung Wook; Clark, Tyson A.; Schweitzer, Anthony C.; Blume, John E.; Wang, Xuning; Darnell, Jennifer C.; Darnell, Robert B.

2008-11-01

Protein-RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.

Super Spy variants implicate flexibility in chaperone action.

PubMed

Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl At; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James Ca

2014-01-01

Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These "Super Spy" variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001.

Super Spy variants implicate flexibility in chaperone action

PubMed Central

Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl AT; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James CA

2014-01-01

Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001 PMID:24497545

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

PubMed Central

Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

2013-01-01

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

Specific cardiolipin binding interferes with labeling of sulfhydryl residues in the adenosine diphosphate/adenosine triphosphate carrier protein from beef heart mitochondria.

PubMed

Beyer, K; Nuscher, B

1996-12-10

The interaction of cardiolipin with the isolated ADP/ATP carrier protein from beef heart mitochondria has been studied by means of the unmasking of a single cysteinyl residue, Cys56, which accompanies the conformational transition of the protein [Leblanc, P., & Clauser, H, (1972) FEBS Lett. 23, 107-113]. The unmasking was monitored by using the static fluorescence of the sulfhydryl reagent N-(1-pyrenyl)maleimide (PYM). The rate of PYM binding that was observed after initiation of the conformational transition by ADP was drastically reduced in the presence of cardiolipin (CL). Phospholipids other than CL were much less effective. It can be shown that the conformational transition and the binding reaction are both affected by CL, although to varying extents. An enhancement of the rate of the ADP-dependent PYM binding was observed upon digestion of the protein bound phospholipid by phospholipase A2. The phospholipase treatment also led to an increased ADP-independent PYM binding, thus indicating that the ADP control of the carrier transition was gradually lost. The ADP control could be fully restored through the addition of CL, provided that the phospholipase incubation had been terminated after approximately 1 h. These results will be discussed in relation to an earlier report of tight cardiolipin binding [Beyer, K., & Klingenberg, M. (1985) Biochemistry 24, 3821-3826] and to current structural models of the ADP/ATP carrier protein.

Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA.

PubMed

Kim, Sanghyun; Zbaida, David; Elbaum, Michael; Leh, Hervé; Nogues, Claude; Buckle, Malcolm

2015-07-27

VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

Giuliani, Sarah E; Frank, Ashley M; Corgliano, Danielle M

Abstract Background: Transporter proteins are one of an organism s primary interfaces with the environment. The expressed set of transporters mediates cellular metabolic capabilities and influences signal transduction pathways and regulatory networks. The functional annotation of most transporters is currently limited to general classification into families. The development of capabilities to map ligands with specific transporters would improve our knowledge of the function of these proteins, improve the annotation of related genomes, and facilitate predictions for their role in cellular responses to environmental changes. Results: To improve the utility of the functional annotation for ABC transporters, we expressed and purifiedmore » the set of solute binding proteins from Rhodopseudomonas palustris and characterized their ligand-binding specificity. Our approach utilized ligand libraries consisting of environmental and cellular metabolic compounds, and fluorescence thermal shift based high throughput ligand binding screens. This process resulted in the identification of specific binding ligands for approximately 64% of the purified and screened proteins. The collection of binding ligands is representative of common functionalities associated with many bacterial organisms as well as specific capabilities linked to the ecological niche occupied by R. palustris. Conclusion: The functional screen identified specific ligands that bound to ABC transporter periplasmic binding subunits from R. palustris. These assignments provide unique insight for the metabolic capabilities of this organism and are consistent with the ecological niche of strain isolation. This functional insight can be used to improve the annotation of related organisms and provides a route to evaluate the evolution of this important and diverse group of transporter proteins.« less

Cytoadhesion to gC1qR through Plasmodium falciparum Erythrocyte Membrane Protein 1 in Severe Malaria

PubMed Central

Magallón-Tejada, Ariel; Machevo, Sónia; Cisteró, Pau; Lavstsen, Thomas; Aide, Pedro; Jiménez, Alfons; Turner, Louise; Gupta, Himanshu; De Las Salas, Briegel; Mandomando, Inacio; Wang, Christian W.; Petersen, Jens E. V.; Muñoz, Jose; Gascón, Joaquim; Macete, Eusebio; Alonso, Pedro L.; Chitnis, Chetan E.

2016-01-01

Cytoadhesion of Plasmodium falciparum infected erythrocytes to gC1qR has been associated with severe malaria, but the parasite ligand involved is currently unknown. To assess if binding to gC1qR is mediated through the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, we analyzed by static binding assays and qPCR the cytoadhesion and var gene transcriptional profile of 86 P. falciparum isolates from Mozambican children with severe and uncomplicated malaria, as well as of a P. falciparum 3D7 line selected for binding to gC1qR (Pf3D7gC1qR). Transcript levels of DC8 correlated positively with cytoadhesion to gC1qR (rho = 0.287, P = 0.007), were higher in isolates from children with severe anemia than with uncomplicated malaria, as well as in isolates from Europeans presenting a first episode of malaria (n = 21) than Mozambican adults (n = 25), and were associated with an increased IgG recognition of infected erythrocytes by flow cytometry. Pf3D7gC1qR overexpressed the DC8 type PFD0020c (5.3-fold transcript levels relative to Seryl-tRNA-synthetase gene) compared to the unselected line (0.001-fold). DBLβ12 from PFD0020c bound to gC1qR in ELISA-based binding assays and polyclonal antibodies against this domain were able to inhibit binding to gC1qR of Pf3D7gC1qR and four Mozambican P. falciparum isolates by 50%. Our results show that DC8-type PfEMP1s mediate binding to gC1qR through conserved surface epitopes in DBLβ12 domain which can be inhibited by strain-transcending functional antibodies. This study supports a key role for gC1qR in malaria-associated endovascular pathogenesis and suggests the feasibility of designing interventions against severe malaria targeting this specific interaction. PMID:27835682

Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen.

PubMed

Yadav, Ashok Kumar; Tyagi, Ashish; Kaushik, Jai Kumar; Saklani, Asha Chandola; Grover, Sunita; Batish, Virender Kumar

2013-12-14

Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored. Copyright © 2013 Elsevier GmbH. All rights reserved.

Effects of two novel amino acid substitutions on the penicillin binding properties of the PBP5 C‑terminal from Enterococcus faecium.

PubMed

Zhou, Chengjiang; Niu, Haiying; Yu, Hui; Zhou, Lishe; Wang, Zhanli

2015-10-01

The low‑affinity penicillin‑binding protein (PBP)5 is responsible for resistance to β‑lactam antibiotics in Enterococcus faecium. (E. faecium). In order to evaluate more fully the potential of this species for the development of resistance to β-lactam antibiotics, the present study aimed to examine the extent of penicillin-binding protein (PBP) variations in a collection of clinical E. faecium isolates. In the present study, the C‑terminal domain of PBP5 (PBP5‑CD) of 13 penicillin‑resistant clinical isolates of E. faecium were sequenced and the correlation between penicillin resistance and particular amino acid changes were analyzed. The present study identified for the first time, to the best of our knowledge, two novel substitutions (Tyr460Phe and Ala462Thr or Val462Thr) of E. faecium PBP5‑CD. The covalent interaction between penicillin and PBP5‑CD was also investigated using homology modeling and molecular docking methods. The theoretical calculation revealed that Phe460 and Thr462 were involved in penicillin binding, suggesting that substitutions at these positions exert effects on the affinity for penicillin, and this increased affinity translates into lower resistance in vitro.

Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing; Progress report, June 1, 1990--May 31, 1993

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

Richardson, C.C.

1993-12-31

This project focuses on the DNA polymerase (gene 5 protein) of phage T7 for use in DNA sequence analysis. Gene 5 protein interacts with accessory proteins to acquire properties essential for DNA replication. One goal is to understand these interactions in order to modify the proteins for use in DNA sequencing. E. coli thioredoxin, binds to gene 5 protein and clamps it to a primer-template. They have analyzed the binding of gene 5 protein-thioredoxin to primer-templates and have defined the optimal conditions to form an extremely stable complex with a dNTP in the polymerase catalytic site. The spatial proximity ofmore » these components has been determined using fluorescence emission anisotropy. The T7 DNA binding protein, the gene 2.5 protein, interacts with gene 5 protein and gene 4 protein to increase processivity and primer synthesis, respectively. Mutant gene 2.5 proteins have been isolated that do not interact with T7 DNA polymerase and can not support T7 growth. The nucleotide binding site of the T7 helicase has been identified and mutations affecting the site provide information on how the hydrolysis of NTPs fuel its unidirectional translocation. The sequence, GTC, has been shown to be necessary and sufficient for recognition by the T7 primase. The T7 gene 5.5 protein interacts with the E. coli nucleoid protein, H-NS, and also overcomes the phage {lambda} rex restriction system.« less

Proteomic Analysis of an α7 Nicotinic Acetylcholine Receptor Interactome

PubMed Central

Paulo, Joao A.; Brucker, William J.; Hawrot, Edward

2009-01-01

The α7 nicotinic acetylcholine receptor (nAChR) is well established as the principal high-affinity α-bungarotoxin-binding protein in the mammalian brain. We isolated carbachol-sensitive α-bungarotoxin-binding complexes from total mouse brain tissue by affinity immobilization followed by selective elution, and these proteins were fractionated by SDS-PAGE. The proteins in subdivided gel lane segments were tryptically digested, and the resulting peptides were analyzed by standard mass spectrometry. We identified 55 proteins in wild-type samples that were not present in comparable brain samples from α7 nAChR knockout mice that had been processed in a parallel fashion. Many of these 55 proteins are novel proteomic candidates for interaction partners of the α7 nAChR, and many are associated with multiple signaling pathways that may be implicated in α7 function in the central nervous system. The newly identified potential protein interactions, together with the general methodology that we introduce for α-bungarotoxin-binding protein complexes, form a new platform for many interesting follow-up studies aimed at elucidating the physiological role of neuronal α7 nAChRs. PMID:19714875

Rapid discovery of protein interactions by cell-free protein technologies.

PubMed

He, M; Taussig, M J

2007-11-01

Cell-free transcription and translation provides an open, controllable environment for production of correctly folded, soluble proteins and allows the rapid generation of proteins from DNA without the need for cloning. Thus it is becoming an increasingly attractive alternative to conventional in vivo expression systems, especially when parallel expression of multiple proteins is required. Through novel design and exploitation, powerful cell-free technologies of ribosome display and protein in situ arrays have been developed for in vitro production and isolation of protein-binding molecules from large libraries. These technologies can be combined for rapid detection of protein interactions.

Effect of inclusion of hydroxycinnamic and chlorogenic acids from green coffee bean in β-cyclodextrin on their interactions with whey, egg white and soy protein isolates.

PubMed

Budryn, Grażyna; Pałecz, Bartłomiej; Rachwał-Rosiak, Danuta; Oracz, Joanna; Zaczyńska, Donata; Belica, Sylwia; Navarro-González, Inmaculada; Meseguer, Josefina María Vegara; Pérez-Sánchez, Horacio

2015-02-01

The aim of the study was to characterise the interactions of hydroxycinnamic and chlorogenic acids (CHAs) from green coffee, with isolates of proteins from egg white (EWP), whey (WPC) and soy (SPI), depending on pH and temperature. The binding degree was determined by liquid chromatography coupled to a diode array detector and an ultrahigh resolution hybrid quadruple-time-of-flight mass spectrometer with ESI source (LC-QTOF-MS/MS). As a result of binding, the concentration of CHAs in proteins ranged from 9.44-12.2, 11.8-13.1 and 12.1-14.4g/100g for SPI, WPC and EWP, respectively. Thermodynamic parameters of protein-ligand interactions were determined by isothermal titration calorimetry (ITC) and energetics of interactions at the atomic level by molecular modelling. The amount of CHAs released during proteolytic digestion was in the range 0.33-2.67g/100g. Inclusion of CHAs with β-cyclodextrin strongly limited these interactions to a level of 0.03-0.06g/100g. Copyright © 2014 Elsevier Ltd. All rights reserved.

Recent insights into the biological functions of liver fatty acid binding protein 1

PubMed Central

Wang, GuQi; Bonkovsky, Herbert L.; de Lemos, Andrew; Burczynski, Frank J.

2015-01-01

Over four decades have passed since liver fatty acid binding protein (FABP)1 was first isolated. There are few protein families for which most of the complete tertiary structures, binding properties, and tissue occurrences are described in such detail and yet new functions are being uncovered for this protein. FABP1 is known to be critical for fatty acid uptake and intracellular transport and also has an important role in regulating lipid metabolism and cellular signaling pathways. FABP1 is an important endogenous cytoprotectant, minimizing hepatocyte oxidative damage and interfering with ischemia-reperfusion and other hepatic injuries. The protein may be targeted for metabolic activation through the cross-talk among many transcriptional factors and their activating ligands. Deficiency or malfunction of FABP1 has been reported in several diseases. FABP1 also influences cell proliferation during liver regeneration and may be considered as a prognostic factor for hepatic surgery. FABP1 binds and modulates the action of many molecules such as fatty acids, heme, and other metalloporphyrins. The ability to bind heme is another cytoprotective property and one that deserves closer investigation. The role of FABP1 in substrate availability and in protection from oxidative stress suggests that FABP1 plays a pivotal role during intracellular bacterial/viral infections by reducing inflammation and the adverse effects of starvation (energy deficiency). PMID:26443794

Protein-ligand binding free energy estimation using molecular mechanics and continuum electrostatics. Application to HIV-1 protease inhibitors

NASA Astrophysics Data System (ADS)

Zoete, V.; Michielin, O.; Karplus, M.

2003-12-01

A method is proposed for the estimation of absolute binding free energy of interaction between proteins and ligands. Conformational sampling of the protein-ligand complex is performed by molecular dynamics (MD) in vacuo and the solvent effect is calculated a posteriori by solving the Poisson or the Poisson-Boltzmann equation for selected frames of the trajectory. The binding free energy is written as a linear combination of the buried surface upon complexation, SAS bur, the electrostatic interaction energy between the ligand and the protein, Eelec, and the difference of the solvation free energies of the complex and the isolated ligand and protein, ΔGsolv. The method uses the buried surface upon complexation to account for the non-polar contribution to the binding free energy because it is less sensitive to the details of the structure than the van der Waals interaction energy. The parameters of the method are developed for a training set of 16 HIV-1 protease-inhibitor complexes of known 3D structure. A correlation coefficient of 0.91 was obtained with an unsigned mean error of 0.8 kcal/mol. When applied to a set of 25 HIV-1 protease-inhibitor complexes of unknown 3D structures, the method provides a satisfactory correlation between the calculated binding free energy and the experimental pIC 50 without reparametrization.

COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein.

PubMed Central

Avram, Dorina; Fields, Andrew; Senawong, Thanaset; Topark-Ngarm, Acharawan; Leid, Mark

2002-01-01

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 [CTIP1/Evi9/B cell leukaemia (Bcl) l1a and CTIP2/Bcl11b respectively] are highly related C(2)H(2) zinc finger proteins that are abundantly expressed in brain and the immune system, and are associated with immune system malignancies. A selection procedure was employed to isolate high-affinity DNA binding sites for CTIP1. The core binding site on DNA identified in these studies, 5'-GGCCGG-3' (upper strand), is highly related to the canonical GC box and was bound by a CTIP1 oligomeric complex(es) in vitro. Furthermore, both CTIP1 and CTIP2 repressed transcription of a reporter gene harbouring a multimerized CTIP binding site, and this repression was neither reversed by trichostatin A (an inhibitor of known class I and II histone deacetylases) nor stimulated by co-transfection of a COUP-TF family member. These results demonstrate that CTIP1 is a sequence-specific DNA binding protein and a bona fide transcriptional repressor that is capable of functioning independently of COUP-TF family members. These findings may be relevant to the physiological and/or pathological action(s) of CTIPs in cells that do not express COUP-TF family members, such as cells of the haematopoietic and immune systems. PMID:12196208

Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq

PubMed Central

Lefrançois, Philippe; Gallagher, Jennifer E. G.; Snyder, Michael

2016-01-01

Transcription factors influence gene expression through their ability to bind DNA at specific regulatory elements. Specific DNA-protein interactions can be isolated through the chromatin immunoprecipitation (ChIP) procedure, in which DNA fragments bound by the protein of interest are recovered. ChIP is followed by high-throughput DNA sequencing (Seq) to determine the genomic provenance of ChIP DNA fragments and their relative abundance in the sample. This chapter describes a ChIP-Seq strategy adapted for budding yeast to enable the genome-wide characterization of binding sites of transcription factors (TFs) and other DNA-binding proteins in an efficient and cost-effective way. Yeast strains with epitope-tagged TFs are most commonly used for ChIP-Seq, along with their matching untagged control strains. The initial step of ChIP involves the cross-linking of DNA and proteins. Next, yeast cells are lysed and sonicated to shear chromatin into smaller fragments. An antibody against an epitope-tagged TF is used to pull down chromatin complexes containing DNA and the TF of interest. DNA is then purified and proteins degraded. Specific barcoded adapters for multiplex DNA sequencing are ligated to ChIP DNA. Short DNA sequence reads (28–36 base pairs) are parsed according to the barcode and aligned against the yeast reference genome, thus generating a nucleotide-resolution map of transcription factor-binding sites and their occupancy. PMID:25213249

Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-beta but interacts differently with RanBP1.

PubMed Central

Deane, R; Schäfer, W; Zimmermann, H P; Mueller, L; Görlich, D; Prehn, S; Ponstingl, H; Bischoff, F R

1997-01-01

We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5. This protein is related to importin-beta, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport. RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait. RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex. Like importin-beta, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange. The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1. RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes. We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-alpha-dependent import of proteins with a classical NLS. PMID:9271386

Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

PubMed

Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

2004-09-01

We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells.

PubMed

Srikrishna, G; Panneerselvam, K; Westphal, V; Abraham, V; Varki, A; Freeze, H H

2001-04-01

We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.

Aspartate aminotransferase is potently inhibited by copper complexes: Exploring copper complex-binding proteome.

PubMed

Jia, Yuqi; Lu, Liping; Yuan, Caixia; Feng, Sisi; Zhu, Miaoli

2017-05-01

Recent researches indicated that a copper complex-binding proteome that potently interacted with copper complexes and then influenced cellular metabolism might exist in organism. In order to explore the copper complex-binding proteome, a copper chelating ion-immobilized affinity chromatography (Cu-IMAC) column and mass spectrometry were used to separate and identify putative Cu-binding proteins in primary rat hepatocytes. A total of 97 putative Cu-binding proteins were isolated and identified. Five higher abundance proteins, aspartate aminotransferase (AST), malate dehydrogenase (MDH), catalase (CAT), calreticulin (CRT) and albumin (Alb) were further purified using a SP-, and (or) Q-Sepharose Fast Flow column. The interaction between the purified proteins and selected 11 copper complexes and CuCl 2 was investigated. The enzymes inhibition tests demonstrated that AST was potently inhibited by copper complexes while MDH and CAT were weakly inhibited. Schiff-based copper complexes 6 and 7 potently inhibited AST with the IC 50 value of 3.6 and 7.2μM, respectively and exhibited better selectivity over MDH and CAT. Fluorescence titration results showed the two complexes tightly bound to AST with binding constant of 3.89×10 6 and 3.73×10 6 M -1 , respectively and a stoichiometry ratio of 1:1. Copper complex 6 was able to enter into HepG2 cells and further inhibit intracellular AST activity. Copyright © 2017 Elsevier Inc. All rights reserved.

TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

PubMed Central

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity. PMID:24349308

Molecular basis and quantitative assessment of TRF1 and TRF2 protein interactions with TIN2 and Apollo peptides.

PubMed

Kalathiya, Umesh; Padariya, Monikaben; Baginski, Maciej

2017-03-01

Shelterin is a six-protein complex (TRF1, TRF2, POT1, RAP1, TIN2, and TPP1) that also functions in smaller subsets in regulation and protection of human telomeres. Two closely related proteins, TRF1 and TRF2, make high-affinity contact directly with double-stranded telomeric DNA and serve as a molecular platform. Protein TIN2 binds to TRF1 and TRF2 dimer-forming domains, whereas Apollo makes interaction only with TRF2. To elucidate the molecular basis of these interactions, we employed molecular dynamics (MD) simulations of TRF1 TRFH -TIN2 TBM and TRF2 TRFH -TIN2 TBM /Apollo TBM complexes and of the isolated proteins. MD enabled a structural and dynamical comparison of protein-peptide complexes including H-bond interactions and interfacial residues that may regulate TRF protein binding to the given peptides, especially focusing on interactions described in crystallographic data. Residues with a selective function in both TRF1 TRFH and TRF2 TRFH and forming a stable hydrogen bond network with TIN2 TBM or Apollo TBM peptides were traced. Our study revealed that TIN2 TBM forms a well-defined binding mode with TRF1 TRFH as compared to TRF2 TRFH , and that the binding pocket of TIN2 TBM is deeper for TRF2 TRFH protein than Apollo TBM . The MD data provide a basis for the reinterpretation of mutational data obtained in crystallographic work for the TRF proteins. Together, the previously determined X-ray structure and our MD provide a detailed view of the TRF-peptide binding mode and the structure of TRF1/2 binding pockets. Particular TRF-peptide interactions are very specific for the formation of each protein-peptide complex, identifying TRF proteins as potential targets for the design of inhibitors/drugs modulating telomere machinery for anticancer therapy.

Structure of the buffalo secretory signalling glycoprotein at 2.8 Å resolution

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

Ethayathulla, Abdul S.; Srivastava, Devendra B.; Kumar, Janesh

2007-04-01

The crystal structure of a signalling glycoprotein isolated from buffalo dry secretions (SPB-40) has been determined at 2.8 Å resolution. Two unique residues, Tyr120 and Glu269, found in SPB-40 distort the shape of the sugar-binding groove considerably. The water structure in the groove is also different. The conformations of three flexible loops, His188–His197, Phe202–Arg212 and Tyr244–Pro260, also differ from those found in other structurally similar proteins. The crystal structure of a 40 kDa signalling glycoprotein from buffalo (SPB-40) has been determined at 2.8 Å resolution. SPB-40 acts as a protective signalling factor by binding to viable cells during the earlymore » phase of involution, during which extensive tissue remodelling occurs. It was isolated from the dry secretions of Murrah buffalo. It was purified and crystallized using the hanging-drop vapour-diffusion method with 19% ethanol as the precipitant. The protein was also cloned and its complete nucleotide and amino-acid sequences were determined. When compared with the sequences of other members of the family, the sequence of SPB-40 revealed two very important mutations in the sugar-binding region, in which Tyr120 changed to Trp120 and Glu269 changed to Trp269. The structure showed a significant distortion in the shape of the sugar-binding groove. The water structure in the groove is also drastically altered. The folding of the protein chain in the flexible region comprising segments His188–His197, Phe202–Arg212 and Tyr244–Pro260 shows large variations when compared with other proteins of the family.« less

Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70.

PubMed

Nuttall, Stewart D; Krishnan, Usha V; Doughty, Larissa; Pearson, Kylie; Ryan, Michael T; Hoogenraad, Nicholas J; Hattarki, Meghan; Carmichael, Jennifer A; Irving, Robert A; Hudson, Peter J

2003-09-01

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

SARNAclust: Semi-automatic detection of RNA protein binding motifs from immunoprecipitation data

PubMed Central

Dotu, Ivan; Adamson, Scott I.; Coleman, Benjamin; Fournier, Cyril; Ricart-Altimiras, Emma; Eyras, Eduardo

2018-01-01

RNA-protein binding is critical to gene regulation, controlling fundamental processes including splicing, translation, localization and stability, and aberrant RNA-protein interactions are known to play a role in a wide variety of diseases. However, molecular understanding of RNA-protein interactions remains limited; in particular, identification of RNA motifs that bind proteins has long been challenging, especially when such motifs depend on both sequence and structure. Moreover, although RNA binding proteins (RBPs) often contain more than one binding domain, algorithms capable of identifying more than one binding motif simultaneously have not been developed. In this paper we present a novel pipeline to determine binding peaks in crosslinking immunoprecipitation (CLIP) data, to discover multiple possible RNA sequence/structure motifs among them, and to experimentally validate such motifs. At the core is a new semi-automatic algorithm SARNAclust, the first unsupervised method to identify and deconvolve multiple sequence/structure motifs simultaneously. SARNAclust computes similarity between sequence/structure objects using a graph kernel, providing the ability to isolate the impact of specific features through the bulge graph formalism. Application of SARNAclust to synthetic data shows its capability of clustering 5 motifs at once with a V-measure value of over 0.95, while GraphClust achieves only a V-measure of 0.083 and RNAcontext cannot detect any of the motifs. When applied to existing eCLIP sets, SARNAclust finds known motifs for SLBP and HNRNPC and novel motifs for several other RBPs such as AGGF1, AKAP8L and ILF3. We demonstrate an experimental validation protocol, a targeted Bind-n-Seq-like high-throughput sequencing approach that relies on RNA inverse folding for oligo pool design, that can validate the components within the SLBP motif. Finally, we use this protocol to experimentally interrogate the SARNAclust motif predictions for protein ILF3. Our results support a newly identified partially double-stranded UUUUUGAGA motif similar to that known for the splicing factor HNRNPC. PMID:29596423

Isolation of monomeric photosystem II that retains the subunit PsbS.

PubMed

Haniewicz, Patrycja; De Sanctis, Daniele; Büchel, Claudia; Schröder, Wolfgang P; Loi, Maria Cecilia; Kieselbach, Thomas; Bochtler, Matthias; Piano, Dario

2013-12-01

Photosystem II has been purified from a transplastomic strain of Nicotiana tabacum according to two different protocols. Using the procedure described in Piano et al. (Photosynth Res 106:221-226, 2010) it was possible to isolate highly active PSII composed of monomers and dimers but depleted in their PsbS protein content. A "milder" procedure than the protocol reported by Fey et al. (Biochim Biophys Acta 1777:1501-1509, 2008) led to almost exclusively monomeric PSII complexes which in part still bind the PsbS protein. This finding might support a role for PSII monomers in higher plants.

Retinal-specific ATP-binding cassette transporter (ABCR/ABCA4) is expressed at the choroid plexus in rat brain.

PubMed

Bhongsatiern, Jiraganya; Ohtsuki, Sumio; Tachikawa, Masanori; Hori, Satoko; Terasaki, Tetsuya

2005-03-01

ATP-binding cassette (ABC) transporter A4 is a member of the ABC transporter subfamily A which has been reported to be exclusively expressed in the retina. In contrast, a previous report has suggested a possible relationship between ABCA4 and CNS function. The purpose of the present study was to investigate the localization of ABCA4 mRNA and protein in rat brain. In situ hybridization analysis revealed that ABCA4 mRNA was localized in the lateral ventricles. RT-PCR analysis detected ABCA4 mRNA in isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells (TR-CSFB). Furthermore, ABCA4 protein was also detected in the isolated rat choroid plexus at about 250 kDa by western blot analysis, and its apparent molecular size was reduced by N-glycosidase F treatment. These results suggest that glycosylated ABCA4 protein is expressed in rat choroid plexus epithelial cells. ABCA4 may play a role in the function of the blood-cerebrospinal fluid barrier and affect CSF conditions.

Antibody phage display: overview of a powerful technology that has quickly translated to the clinic.

PubMed

Kotlan, Beatrix; Glassy, Mark C

2009-01-01

Antibody-based immunologic reagents are useful for identifying, isolating, or eliminating cells with particular characteristics related to different diseases. Phage display is a highly valuable technique for antibody selection related to this purpose. In brief, a diverse group of antibody genes prepared from a patient or generated in vitro are inserted into a phagemid vector or the phage genome so that when the protein is expressed, it becomes anchored on the surface of the phage by fusion to a coat protein. A diverse library of recombinant antibodies is generated in this way and can then be exposed or panned on the antigen of interest, typically, this being a molecule associated with a particular pathological condition. Phage that carry proteins or peptides bind preferentially to the target and can thus be isolated from the library. The viruses that are recovered in this way also carry the gene for the binding moiety facilitating its over-expression or manipulation. Recent reviews highlight key milestones in the development of antibody libraries and their screening by phage display, and the impact of these technologies on drug discovery seems assured.

Ascidian sperm glycosylphosphatidylinositol-anchored CRISP-like protein as a binding partner for an allorecognizable sperm receptor on the vitelline coat.

PubMed

Urayama, Satoshi; Harada, Yoshito; Nakagawa, Yoko; Ban, Susumu; Akasaka, Mari; Kawasaki, Nana; Sawada, Hitoshi

2008-08-01

Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain

DOE PAGES

Wu, R.; Wilton, R.; Cuff, M. E.; ...

2017-02-07

The tandem Per-Arnt-Sim (PAS) like sensors are commonly found in signal transduction proteins. The periplasmic solute binding protein (SBP) domains are found ubiquitously and are generally involved in solute transport. These domains are widely observed as parts of separate proteins but not within the same polypeptide chain. We report the structural and biochemical characterization of the extracellular ligand-binding receptor, Dret_0059 from Desulfohalobium retbaense DSM 5692, an organism isolated from the Retba salt lake in Senegal. The structure of Dret_0059 consists of a novel combination of SBP and TPAS sensor domains. The N-terminal region forms an SBP domain and the C-terminalmore » region folds into a tandem PAS-like domain structure. A ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS domain of the TPAS. The differential scanning flourimetry studies in solution support the ligands observed in the crystal structure. There are only two other proteins with this structural architecture in the non-redundant sequence data base and we predict that they too bind the same substrates. There is significant interaction between the SBP and TPAS domains, and it is quite conceivable that the binding of one ligand will have an effect on the binding of the other. Our attempts to remove the ligands bound to the protein during expression were not successful, therefore, it is not clear what the relative affects are. The genomic context of this receptor does not contain any protein components expected for transport function, hence, we suggest that Dret_0059 is likely involved in signal transduction and not in solute transport.« less

Dissecting binding of a β-barrel membrane protein by phage display.

PubMed

Meneghini, Luz M; Tripathi, Sarvind; Woodworth, Marcus A; Majumdar, Sudipta; Poulos, Thomas L; Weiss, Gregory A

2017-07-25

Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal. Here, we apply a new approach for the expedited protein characterization of the TBDT ShuA from Shigella dysenteriae, and elucidate the protein's initial steps during heme-uptake. ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable MP variants for rapid characterization of binding to various ligands. Site-directed mutagenesis studies targeting each extracellular loop region of ShuA demonstrate no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. The results identify a loop susceptible to antibody binding, and also a small molecule motif capable of disrupting ShuA from S. dysenteriae. The approach is generalizable to the dissection of other phage-displayed TBDTs and MPs.

Evolution of RNA-Protein Interactions: Non-Specific Binding Led to RNA Splicing Activity of Fungal Mitochondrial Tyrosyl-tRNA Synthetases

PubMed Central

Lamech, Lilian T.; Mallam, Anna L.; Lambowitz, Alan M.

2014-01-01

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was “fixed” by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins. PMID:25536042

Evolution of RNA-protein interactions: non-specific binding led to RNA splicing activity of fungal mitochondrial tyrosyl-tRNA synthetases.

PubMed

Lamech, Lilian T; Mallam, Anna L; Lambowitz, Alan M

2014-12-01

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was "fixed" by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins.

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage-display

PubMed Central

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K.

2012-01-01

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious and time consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13, the N-terminal Forkhead-associated domain (FHA1) of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be non-functional due to misfolding in the bacterial periplasm. To overcome this limitation, a library of FHA1 variants was constructed by mutagenic PCR and functional variants were isolated after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1-strand was discovered to be essential for phage-display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermal stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20–25 mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage-display. PMID:22985966

Bone Matrix Proteins: Isolation and Characterization of a Novel Cell-binding Keratan Sulfate Proteoglycan (Osteoadherin) from Bovine Bone

PubMed Central

Wendel, Mikael; Sommarin, Yngve; Heinegård, Dick

1998-01-01

A small cell-binding proteoglycan for which we propose the name osteoadherin was extracted from bovine bone with guanidine hydrochloride–containing EDTA. It was purified to homogeneity using a combination of ion-exchange chromatography, hydroxyapatite chromatography, and gel filtration. The Mr of the proteoglycan was 85,000 as determined by SDS-PAGE. The protein is rich in aspartic acid, glutamic acid, and leucine. Two internal octapeptides from the proteoglycan contained the sequences Glu-Ile-Asn-Leu-Ser-His-Asn-Lys and Arg-Asp-Leu-Tyr-Phe-Asn-Lys-Ile. These sequences are not previously described, and support the notion that osteoadherin belongs to the family of leucine-rich repeat proteins. A monospecific antiserum was raised in rabbits. An enzyme-linked immunosorbent assay was developed, and showed the osteoadherin content of bone extracts to be 0.4 mg/g of tissue wet weight, whereas none was found in extracts of various other bovine tissues. Metabolic labeling of primary bovine osteoblasts followed by immunoprecipitation showed the cells to synthesize and secrete the proteoglycan. Digesting the immunoprecipitated osteoadherin with N-glycosidase reduced its apparent size to 47 kD, thus showing the presence of several N-linked oligosaccharides. Digestion with keratanase indicated some of the oligosaccharides to be extended to keratan sulfate chains. In immunohistochemical studies of the bovine fetal rib growth plate, osteoadherin was exclusively identified in the primary bone spongiosa. Osteoadherin binds to hydroxyapatite. A potential function of this proteoglycan is to bind cells, since we showed it to be as efficient as fibronectin in promoting osteoblast attachment in vitro. The binding appears to be mediated by the integrin αvβ3, since this was the only integrin isolated by osteoadherin affinity chromatography of surface-iodinated osteoblast extracts. PMID:9566981

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage display.

PubMed

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K

2012-11-23

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious, and time-consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13 the N-terminal Forkhead-associated (FHA1) domain of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be nonfunctional due to misfolding in the bacterial periplasm. To overcome this limitation, we constructed a library of FHA1 variants by mutagenic PCR and isolated functional variants after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1 strand was discovered to be essential for phage display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermally stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20-25mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage display. Copyright © 2012 Elsevier Ltd. All rights reserved.

Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling*

PubMed Central

McGarvey, Jennifer C.; Xiao, Kunhong; Bowman, Shanna L.; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W. Bruce; Ardura, Juan A.; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A.; Friedman, Peter A.

2016-01-01

The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860

Phage Display Derived IgNAR V Region Binding Domains for Therapeutic Development.

PubMed

Ubah, Obinna C; Barelle, Caroline J; Buschhaus, Magdalena J; Porter, Andrew J

2016-01-01

Phage display technology has revolutionized the science of drug discovery by transforming the generation and manipulation of ligands, such as antibody fragments, enzymes, and peptides. The basis of this technology is the expression of recombinant proteins or peptides fused to a phage coat protein, and subsequent isolation of ligands based on a variety of catalytic, physicochemical/binding kinetic and/or biological characteristics. An incredible number of diagnostic and therapeutic domains have been successfully isolated using phage display technology. The variable domain of the New Antigen Receptors (VNAR) found in cartilaginous fish, is also amenable to phage display selection. Whilst not an antibody, VNARs are unquestionable the oldest (450 million years), and smallest antigen binding, single-domains so far identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established, enhancing our understanding of the evolutionary origins of humoral immunity and the unusual but divergent ancestry of the VNARs themselves. VNARs exhibit remarkable physicochemical properties, such as small size, stability in extreme conditions, solubility, molecular flexibility, high affinity and selectivity for target. The purpose of this review is to illustrate the important role phage display has played in the isolation and characterization of potent therapeutic and diagnostic VNAR domains. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Streptococcus pneumoniae PspC Subgroup Prevalence in Invasive Disease and Differences in Contribution to Complement Evasion.

PubMed

van der Maten, Erika; van den Broek, Bryan; de Jonge, Marien I; Rensen, Kim J W; Eleveld, Marc J; Zomer, Aldert L; Cremers, Amelieke J H; Ferwerda, Gerben; de Groot, Ronald; Langereis, Jeroen D; van der Flier, Michiel

2018-04-01

The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of Streptococcus pneumoniae isolated from adult patients. pspC deletion mutants and isogenic pspC switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I pspC was far more prevalent in IPD isolates than subgroup II pspC The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition. Copyright © 2018 American Society for Microbiology.

Differences in the Mechanism of the Allosteric L-Rhamnose Responses of the AraC/XylS Family Transcription Activators RhaS and RhaR

PubMed Central

Kolin, Ana; Balasubramaniam, Vinitha; Skredenske, Jeff; Wickstrum, Jason; Egan, Susan M.

2008-01-01

SUMMARY Proteins in the largest subset of AraC/XylS family transcription activators, including RhaS and RhaR, have C-terminal domains (CTDs) that mediate DNA-binding and transcription activation, and N-terminal domains (NTDs) that mediate dimerization and effector binding. The mechanism of the allosteric effector response in this family has been identified only for AraC. Here, we investigated the mechanism by which RhaS and RhaR respond to their effector, L-rhamnose. Unlike AraC, N-terminal truncations suggested that RhaS and RhaR don’t use an N-terminal arm to inhibit activity in the absence of effector. We used random mutagenesis to isolate RhaS and RhaR variants with enhanced activation in the absence of L-rhamnose. NTD substitutions largely clustered around the predicted L-rhamnose-binding pockets, suggesting that they mimic the structural outcome of effector binding to the wild-type proteins. RhaS-CTD substitutions clustered in the first HTH motif, and suggested that L-rhamnose induces improved DNA binding. In contrast, RhaR-CTD substitutions clustered at a single residue in the second HTH motif, at a position consistent with improved RNAP contacts. We propose separate allosteric mechanisms for the two proteins: Without L-rhamnose, RhaS doesn’t effectively bind DNA while RhaR doesn’t effectively contact RNAP. Upon L-rhamnose binding, both proteins undergo structural changes that enable transcription activation. PMID:18366439

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

PubMed

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

1989-01-16

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

Production of recombinant scFv against p24 of human immunodeficiency virus type 1 by phage display technology.

PubMed

Mohammadzadeh, Sara; Rajabibazl, Masoumeh; Fourozandeh, Mehdi; Rasaee, Mohammad Javad; Rahbarizadeh, Fatemeh; Mohammadi, Mohammad

2014-02-01

Phage display has a fundamental role in protein isolation and engineering. Isolated proteins produced with this method can be modified for specific binding and affinity. P24 is the most produced protein during human immune deficiency virus (HIV) replication; especially in the early steps of HIV-1 infection, its evaluation may have diagnostic values. To test the HIV-1 infection, p24 antigen assay appears to be a very promising alternative to RNA assays. In this study, we have generated a recombinant mouse single chain antibody fragment against p24 of the HIV-1 with the use of phage display technology. After isolation of antibody variable-region (V) gene of B cells extracted from the spleen of an immunized mouse, a library of single chain Fv fragments (scFv) was constructed. The library was used in a series of bio-panning processes against recombinant p24 protein expressed from Escherichia coli. The isolated scFv antibody specifically recognizes the HIV-1 capsid protein p24. The affinity constant of the isolated scFv antibody (MF85) was found to be 2×10(-9) M. Our studies showed that the MF85 scFV antibody has similar properties as that of monoclonal antibodies produced by the hybridoma technology.

Acetylcholine, ATP, and proteoglycan are common to synaptic vesicles isolated from the electric organs of electric eel and electric catfish as well as from rat diaphragm.

PubMed

Volknandt, W; Zimmermann, H

1986-11-01

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.

BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax.

PubMed

Wilfling, F; Weber, A; Potthoff, S; Vögtle, F-N; Meisinger, C; Paschen, S A; Häcker, G

2012-08-01

During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.

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

PubMed Central

Heyma, P; Harrison, L C

1984-01-01

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

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

PubMed

Kadamur, Ganesh; Ross, Elliott M

2016-05-20

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

PubMed Central

Kadamur, Ganesh

2016-01-01

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. PMID:27002154

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-02-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed Central

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-01-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. PMID:9927419

[Biochemical characteristics and antigenic structures of Chlamydia].

PubMed

Puy, H; Fuentes, V; Eb, F; Orfila, J

1989-01-01

New biotechnology in immunology and molecular biology has enabled the identification and definition of the structure of glycolipids and especially membrane proteins of Chlamydia. Chlamydia antigen lipopolysaccharide, major outer membrane protein, protein 74 kDa, eukaryotic cell binding protein and cysteine rich proteins are all carriers of antigenic determinants, genus, species or type specific. They are very usefull for diagnosis of Chlamydial infections and epidemiological studies. These membranous antigens have an important role in the pathogenesis of these bacteries. Finally these studies have contributed to the isolation of a new species: C. pneumoniae (TWAR strains).

β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae).

PubMed

Fruttero, Leonardo L; Demartini, Diogo R; Rubiolo, Edilberto R; Carlini, Célia R; Canavoso, Lilián E

2014-09-01

Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [(3)H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-(3)H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.

PubMed

Marshall, Owen J; Southall, Tony D; Cheetham, Seth W; Brand, Andrea H

2016-09-01

This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.

Trapping of the Enoyl-Acyl Carrier Protein Reductase–Acyl Carrier Protein Interaction

PubMed Central

Tallorin, Lorillee; Finzel, Kara; Nguyen, Quynh G.; Beld, Joris; La Clair, James J.; Burkart, Michael D.

2016-01-01

An ideal target for metabolic engineering, fatty acid biosynthesis remains poorly understood on a molecular level. These carrier protein-dependent pathways require fundamental protein–protein interactions to guide reactivity and processivity, and their control has become one of the major hurdles in successfully adapting these biological machines. Our laboratory has developed methods to prepare acyl carrier proteins (ACPs) loaded with substrate mimetics and cross-linkers to visualize and trap interactions with partner enzymes, and we continue to expand the tools for studying these pathways. We now describe application of the slow-onset, tight-binding inhibitor triclosan to explore the interactions between the type II fatty acid ACP from Escherichia coli, AcpP, and its corresponding enoyl-ACP reductase, FabI. We show that the AcpP–triclosan complex demonstrates nM binding, inhibits in vitro activity, and can be used to isolate FabI in complex proteomes. PMID:26938266

bfr1+, a novel gene of Schizosaccharomyces pombe which confers brefeldin A resistance, is structurally related to the ATP-binding cassette superfamily.

PubMed Central

Nagao, K; Taguchi, Y; Arioka, M; Kadokura, H; Takatsuki, A; Yoda, K; Yamasaki, M

1995-01-01

We have isolated a Schizosaccharomyces pombe gene, bfr1+, which on a multicopy plasmid vector, pDB248', confers resistance to brefeldin A (BFA), an inhibitor of intracellular protein transport. This gene encodes a novel protein of 1,531 amino acids with an intramolecular duplicated structure, each half containing a single ATP-binding consensus sequence and a set of six transmembrane sequences. This structural characteristic of bfr1+ protein resembles that of mammalian P-glycoprotein, which, by exporting a variety of anticancer drugs, has been shown to be responsible for multidrug resistance in tumor cells. Consistent with this is that S. pombe cells harboring bfr1+ on pDB248' are resistant to actinomycin D, cerulenin, and cytochalasin B, as well as to BFA. The relative positions of the ATP-binding sequences and the clusters of transmembrane sequences within the bfr1+ protein are, however, transposed in comparison with those in P-glycoprotein; the bfr1+ protein has N-terminal ATP-binding sequence followed by transmembrane segments in each half of the molecule. The bfr1+ protein exhibited significant homology in primary and secondary structures with two recently identified multidrug resistance gene products of Saccharomyces cerevisiae, Snq2 and Sts1/Pdr5/Ydr1. The bfr1+ gene is not essential for cell growth or mating, but a delta bfr1 mutant exhibited hypersensitivity to BFA. We propose that the bfr1+ protein is another member of the ATP-binding cassette superfamily and serves as an efflux pump of various antibiotics. PMID:7883711

Two Multidrug-Resistant Clinical Isolates of Bacteroides fragilis Carry a Novel Metronidazole Resistance nim Gene (nimJ)

PubMed Central

Veeranagouda, Yaligara; Hsi, Justin; Meggersee, Rosemary; Abratt, Valerie; Wexler, Hannah M.

2013-01-01

Two multidrug-resistant Bacteroides fragilis clinical isolates contain and express a novel nim gene, nimJ, that is not recognized by the “universal” nim primers and can confer increased resistance to metronidazole when introduced into a susceptible strain on a multicopy plasmid. HMW615, an appendiceal isolate, contains at least two copies of nimJ on its genome, while HMW616, an isolate from a patient with sepsis, contains one genomic copy of nimJ. B. fragilis NimJ is phylogenetically closer to Prevotella baroniae NimI and Clostridium botulinum NimA than to the other known Bacteroides Nim proteins. The predicted protein structure of NimJ, based on fold recognition analysis, is consistent with the crystal structures derived for known Nim proteins, and specific amino acid residues important for substrate binding in the active site are conserved. This study demonstrates that the “universal” nim primers will not detect all nim genes with the ability to confer metronidazole resistance, but nimJ alone cannot account for the very high metronidazole MICs of these resistant clinical isolates. PMID:23716049

Adhesion properties of potentially probiotic Lactobacillus kefiri to gastrointestinal mucus.

PubMed

Carasi, Paula; Ambrosis, Nicolás M; De Antoni, Graciela L; Bressollier, Philippe; Urdaci, María C; Serradell, María de los Angeles

2014-02-01

We investigated the mucus-binding properties of aggregating and non-aggregating potentially probiotic strains of kefir-isolated Lactobacillus kefiri, using different substrates. All the strains were able to adhere to commercial gastric mucin (MUCIN) and extracted mucus from small intestine (SIM) and colon (CM). The extraction of surface proteins from bacteria using LiCl or NaOH significantly reduced the adhesion of three selected strains (CIDCA 8348, CIDCA 83115 and JCM 5818); although a significant proportion (up to 50%) of S-layer proteins were not completely eliminated after treatments. The surface (S-layer) protein extracts from all the strains of Lb. kefiri were capable of binding to MUCIN, SIM or CM, and no differences were observed among them. The addition of their own surface protein extract increased adhesion of CIDCA 8348 and 83115 to MUCIN and SIM, meanwhile no changes in adhesion were observed for JCM 5818. None of the seven sugars tested had the ability to inhibit the adhesion of whole bacteria to the three mucus extracts. Noteworthy, the degree of bacterial adhesion reached in the presence of their own surface protein (S-layer) extract decreased to basal levels in the presence of some sugars, suggesting an interaction between the added sugar and the surface proteins. In conclusion, the ability of these food-isolated bacteria to adhere to gastrointestinal mucus becomes an essential issue regarding the biotechnological potentiality of Lb. kefiri for the food industry.

Molecular cloning and subcellular localization of Tektin2-binding protein 1 (Ccdc 172) in rat spermatozoa.

PubMed

Yamaguchi, Airi; Kaneko, Takane; Inai, Tetsuichiro; Iida, Hiroshi

2014-04-01

Tektins (TEKTs) are composed of a family of filament-forming proteins localized in cilia and flagella. Five types of mammalian TEKTs have been reported, all of which have been verified to be present in sperm flagella. TEKT2, which is indispensable for sperm structure, mobility, and fertilization, was present at the periphery of the outer dense fiber (ODF) in the sperm flagella. By yeast two-hybrid screening, we intended to isolate flagellar proteins that could interact with TEKT2, which resulted in the isolation of novel two genes from the mouse testis library, referred as a TEKT2-binding protein 1 (TEKT2BP1) and -protein 2 (TEKT2BP2). In this study, we characterized TEKT2BP1, which is registered as a coiled-coil domain-containing protein 172 (Ccdc172) in the latest database. RT-PCR analysis indicated that TEKT2BP1 was predominantly expressed in rat testis and that its expression was increased after 3 weeks of postnatal development. Immunocytochemical studies discovered that TEKT2BP1 localized in the middle piece of rat spermatozoa, predominantly concentrated at the mitochondria sheath of the flagella. We hypothesize that the TEKT2-TEKT2BP1 complex might be involved in the structural linkage between the ODF and mitochondria in the middle piece of the sperm flagella.

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis.

PubMed

Wilson, Kris; Webster, Scott P; Iredale, John P; Zheng, Xiaozhong; Homer, Natalie Z; Pham, Nhan T; Auer, Manfred; Mole, Damian J

2017-12-15

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis

NASA Astrophysics Data System (ADS)

Wilson, Kris; Webster, Scott P.; Iredale, John P.; Zheng, Xiaozhong; Homer, Natalie Z.; Pham, Nhan T.; Auer, Manfred; Mole, Damian J.

2018-01-01

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

C1q Protein Binds to the Apoptotic Nucleolus and Causes C1 Protease Degradation of Nucleolar Proteins*

PubMed Central

Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

2015-01-01

In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. PMID:26231209

Feeding soy protein isolate and treatment with estradiol have different effects on mammary gland morphology and gene expression in weanling male and female rats

USDA-ARS?s Scientific Manuscript database

Isoflavones are phytochemical components of soy diets that bind weakly to estrogen receptors (ERs). To study potential estrogen-like actions of soy in the mammary gland during early development, we fed weanling male and female Sprague-Dawley rats a semi-purified diet with casein as the sole protein ...

Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora

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

Kumar, Kalari Satish; Ravi Kumar, B.; Siddavattam, Dayananda

2006-07-07

In continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (CDRE) in the -3730 bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45-50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtainedmore » from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17 kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17 kDa protein matched with the regulatory {beta}-subunit of calcineurin (Ca{sup 2+}-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression.« less

The use of phage display in neurobiology.

PubMed

Bradbury, Andrew R M

2010-04-01

Phage display has been extensively used to study protein-protein interactions, receptor- and antibody-binding sites, and immune responses, to modify protein properties, and to select antibodies against a wide range of different antigens. In the format most often used, a polypeptide is displayed on the surface of a filamentous phage by genetic fusion to one of the coat proteins, creating a chimeric coat protein, and coupling phenotype (the protein) to genotype (the gene within). As the gene encoding the chimeric coat protein is packaged within the phage, selection of the phage on the basis of the binding properties of the polypeptide displayed on the surface simultaneously results in the isolation of the gene encoding the polypeptide. This unit describes the background to the technique, and illustrates how it has been applied to a number of different problems, each of which has its neurobiological counterparts. Although this overview concentrates on the use of filamentous phage, which is the most popular platform, other systems are also described. (c) 2010 by John Wiley & Sons, Inc.

SSB-1 of the yeast Saccharomyces cerevisiae is a nucleolar-specific, silver-binding protein that is associated with the snR10 and snR11 small nuclear RNAs

PubMed Central

1990-01-01

SSB-1, the yeast single-strand RNA-binding protein, is demonstrated to be a yeast nucleolar-specific, silver-binding protein. In double-label immunofluorescence microscopy experiments antibodies to two other nucleolar proteins, RNA Pol I 190-kD and fibrillarin, were used to reveal the site of rRNA transcription; i.e., the fibrillar region of the nucleolus. SSB-1 colocalized with fibrillarin in a double-label immunofluorescence mapping experiment to the yeast nucleolus. SSB-1 is located, though, over a wider region of the nucleolus than the transcription site marker. Immunoprecipitations of yeast cell extracts with the SSB-1 antibody reveal that in 150 mM NaCl SSB-1 is bound to two small nuclear RNAs (snRNAs). These yeast snRNAs are snR10 and snR11, with snR10 being predominant. Since snR10 has been implicated in pre-rRNA processing, the association of SSB-1 and snR10 into a nucleolar snRNP particle indicates SSB-1 involvement in rRNA processing as well. Also, another yeast protein, SSB-36-kD, isolated by single- strand DNA chromatography, is shown to bind silver under the conditions used for nucleolar-specific staining. It is, most likely, another yeast nucleolar protein. PMID:2121740

The Gab1 protein is a docking site for multiple proteins involved in signaling by the B cell antigen receptor.

PubMed

Ingham, R J; Holgado-Madruga, M; Siu, C; Wong, A J; Gold, M R

1998-11-13

Gab1 is a member of the docking/scaffolding protein family which includes IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins contain a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites for Src homology 2 (SH2) domain-containing signaling proteins. We show in the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosine phosphorylation of Gab1 correlated with the binding of several SH2-containing signaling proteins to Gab1 including Shc, Grb2, phosphatidylinositol 3-kinase, and the SHP-2 tyrosine phosphatase. Far Western analysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosphorylated Gab1 isolated from activated RAMOS cells. In contrast, the Grb2 SH2 domain did not bind directly to Gab1 but instead to the Shc and SHP-2 associated with Gab1. We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/signaling protein complexes are found in this fraction after BCR engagement. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signaling proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BCR signaling pathways.

Differences in Surface-Exposed Antigen Expression between Helicobacter pylori Strains Isolated from Duodenal Ulcer Patients and from Asymptomatic Subjects

PubMed Central

Thoreson, Ann-Catrin E.; Hamlet, Annika; Çelik, Janet; Byström, Mona; Nyström, Susanne; Olbe, Lars; Svennerholm, Ann-Mari

2000-01-01

We have analyzed possible qualitative and quantitative differences in antigen expression between Helicobacter pylori strains isolated from the antrum and different locations in the duodenum of 21 duodenal ulcer (DU) patients and 20 asymptomatic subjects (AS) by enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA. Almost all antral and duodenal strains grown in vitro expressed the N-acetyl-neuroaminyllactose-binding hemagglutinin, flagellins (subunits FlaA and FlaB), urease, a 26-kDa protein, and a neutrophil-activating protein. In 75% of both the DU patients and the AS, antral H. pylori strains expressed either the blood group antigen Lewis y (Ley) alone or together with the Lex antigen. However, duodenal H. pylori strains of DU patients expressed Ley antigen more frequently than corresponding strains of AS (P < 0.05). Presence of Ley on H. pylori was related to the degree of active duodenitis (P < 0.05). Duodenal H. pylori strains isolated from AS were significantly more often Lewis nontypeable than duodenal strains of DU patients (P < 0.01). Presence of H. pylori blood group antigen-binding adhesin (BabA) was significantly higher on both antral and duodenal strains isolated from DU patients than on corresponding strains isolated from AS (P < 0.05). BabA-positive duodenal H. pylori strains isolated from DU patients were associated with active duodenitis more frequently than corresponding strains isolated from AS (P < 0.01). Infection with H. pylori strains positive for Ley and BabA in the duodenum is associated with development of duodenal ulcer formation. PMID:10970397

Phage display: concept, innovations, applications and future.

PubMed

Pande, Jyoti; Szewczyk, Magdalena M; Grover, Ashok K

2010-01-01

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field. Copyright © 2010 Elsevier Inc. All rights reserved.

Novel method for detection of glycogen in cells

PubMed Central

Segvich, Dyann M; DePaoli-Roach, Anna A; Roach, Peter J

2017-01-01

Abstract Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing glutathione S-transferase, a cMyc eptitope and the Stbd1CBM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions. PMID:28077463

The 5.5 protein of phage T7 inhibits H-NS through interactions with the central oligomerization domain.

PubMed

Ali, Sabrina S; Beckett, Emily; Bae, Sandy Jeehoon; Navarre, William Wiley

2011-09-01

The 5.5 protein (T7p32) of coliphage T7 (5.5(T7)) was shown to bind and inhibit gene silencing by the nucleoid-associated protein H-NS, but the mechanism by which it acts was not understood. The 5.5(T7) protein is insoluble when expressed in Escherichia coli, but we find that 5.5(T7) can be isolated in a soluble form when coexpressed with a truncated version of H-NS followed by subsequent disruption of the complex during anion-exchange chromatography. Association studies reveal that 5.5(T7) binds a region of H-NS (residues 60 to 80) recently found to contain a distinct domain necessary for higher-order H-NS oligomerization. Accordingly, we find that purified 5.5(T7) can disrupt higher-order H-NS-DNA complexes in vitro but does not abolish DNA binding by H-NS per se. Homologues of the 5.5(T7) protein are found exclusively among members of the Autographivirinae that infect enteric bacteria, and despite fairly low sequence conservation, the H-NS binding properties of these proteins are largely conserved. Unexpectedly, we find that the 5.5(T7) protein copurifies with heterogeneous low-molecular-weight RNA, likely tRNA, through several chromatography steps and that this interaction does not require the DNA binding domain of H-NS. The 5.5 proteins utilize a previously undescribed mechanism of H-NS antagonism that further highlights the critical importance that higher-order oligomerization plays in H-NS-mediated gene repression. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

GsCBRLK, a calcium/calmodulin-binding receptor-like kinase, is a positive regulator of plant tolerance to salt and ABA stress.

PubMed

Yang, Liang; Ji, Wei; Zhu, Yanming; Gao, Peng; Li, Yong; Cai, Hua; Bai, Xi; Guo, Dianjing

2010-05-01

Calcium/calmodulin-dependent kinases play vital roles in protein phosphorylation in eukaryotes, yet little is known about the phosphorylation process of calcium/calmodulin-dependent protein kinase and its role in stress signal transduction in plants. A novel plant-specific calcium-dependent calmodulin-binding receptor-like kinase (GsCBRLK) has been isolated from Glycine soja. A subcellular localization study using GFP fusion protein indicated that GsCBRLK is localized in the plasma membrane. Binding assays demonstrated that calmodulin binds to GsCBRLK with an affinity of 25.9 nM in a calcium-dependent manner and the binding motif lies between amino acids 147 to169 within subdomain II of the kinase domain. GsCBRLK undergoes autophosphorylation and Myelin Basis Protein phosphorylation in the presence of calcium. It was also found that calcium/calmodulin positively regulates GsCBRLK kinase activity through direct interaction between the calmodulin-binding domain and calmodulin. So, it is likely that GsCBRLK responds to an environmental stimulus in two ways: by increasing the protein expression level and by regulating its kinase activity through the calcium/calmodulin complex. Furthermore, cold, salinity, drought, and ABA stress induce GsCBRLK gene transcripts. Over-expression of GsCBRLK in transgenic Arabidopsis resulted in enhanced plant tolerance to high salinity and ABA and increased the expression pattern of a number of stress gene markers in response to ABA and high salt. These results identify GsCBRLK as a molecular link between the stress- and ABA-induced calcium/calmodulin signal and gene expression in plant cells.

Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

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

Mikhailov, Victor S.; N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808; Vanarsdall, Adam L.

2008-01-20

DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His{sub 6}-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA andmore » that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates.« less

Dissecting the Binding between Glutamine Synthetase and Its Two Natively Unfolded Protein Inhibitors.

PubMed

Pantoja-Uceda, David; Neira, José L; Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel; Santoro, Jorge

2016-06-21

Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue (IF7) and the 149-residue one (IF17). In this work, we studied both IF7 and IF17 by nuclear magnetic resonance (NMR), and we described their binding to GS by using NMR and biolayer interferometry. We assigned the backbone nuclei of all residues of IF7. Analyses of chemical shifts and the (15)N-{(1)H} NOEs at two field strengths suggest that IF7 region Thr3-Arg13 and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The two-dimensional (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting first with IF7 were Gln6 and Ser27, belonging to those regions that appeared to be ordered in the isolated protein. We also determined the kon values and koff values for the binding of both IF7 and IF17 to GS, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model ([Formula: see text]), (ii) there is a strong electrostatic component in the determined kon, and (iii) the binding is not diffusion-limited.

Functional and probiotic attributes of an indigenous isolate of Lactobacillus plantarum.

PubMed

Kaushik, Jai K; Kumar, Ashutosh; Duary, Raj K; Mohanty, Ashok K; Grover, Sunita; Batish, Virender K

2009-12-01

Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt. An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5-2.0 and toxicity of 1.5-2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of approximately 37% and autoaggregation of approximately 31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4+/-1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus. The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.

Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins.

PubMed

Kutin, Yuri; Srinivas, Vivek; Fritz, Matthieu; Kositzki, Ramona; Shafaat, Hannah S; Birrell, James; Bill, Eckhard; Haumann, Michael; Lubitz, Wolfgang; Högbom, Martin; Griese, Julia J; Cox, Nicholas

2016-09-01

A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of class Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is unclear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of Mn II and Fe II . Using EPR and Mössbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess Mn II promotes heterobimetallic cofactor assembly. In the absence of Fe II , R2c cooperatively binds Mn II at both metal sites, whereas R2lox does not readily bind Mn II at either site. Heterometallic cofactor assembly is favored at substoichiometric Fe II concentrations in R2lox. Fe II and Mn II likely bind to the protein in a stepwise fashion, with Fe II binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of Mn II by Fe II at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular Mn II /Fe II concentrations in the host organisms from which they were isolated. Copyright © 2016 Elsevier Inc. All rights reserved.

Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

PubMed

Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; Mei, Yongjie

2015-04-01

Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein-specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization-related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

PubMed

Hua, Weiwei; Lou, Yimin; Xu, Weiyuan; Cheng, Zhixian; Gong, Xingwen; Huang, Jianying

2016-01-01

Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes.

B61 is a ligand for the ECK receptor protein-tyrosine kinase.

PubMed

Bartley, T D; Hunt, R W; Welcher, A A; Boyle, W J; Parker, V P; Lindberg, R A; Lu, H S; Colombero, A M; Elliott, R L; Guthrie, B A

1994-04-07

A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

Bombyx mori Nucleopolyhedrovirus Encodes a DNA-Binding Protein Capable of Destabilizing Duplex DNA

PubMed Central

Mikhailov, Victor S.; Mikhailova, Alla L.; Iwanaga, Masashi; Gomi, Sumiko; Maeda, Susumu

1998-01-01

A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from Bombyx mori) infected with the B. mori nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no. L33180). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3′→5′ exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication. PMID:9525636

Glycation of whey protein with dextrans of different molar mass: Effect on immunoglobulin E-binding capacity with blood sera obtained from patients with cow milk protein allergy.

PubMed

Xu, Lei; Gong, Yuansheng; Gern, James E; Ikeda, Shinya; Lucey, John A

2018-05-16

A growing concern around the world is the number of people who are suffering from food protein allergies. One potential approach to decrease protein allergenicity is to block IgE-binding epitopes of the protein allergen by attachment of polysaccharides via the Maillard reaction (i.e., glycation). Protein glycation has been extensively studied to modify various functional properties. We wanted to examine whether glycates could reduce IgE binding in patients with cow milk protein allergy and to explore how the size (molar mass; M W ) of the polysaccharide affects this IgE-binding capacity. Glycation was performed using the initial step of the Maillard reaction performed in aqueous solutions. The specific goal of this study was to reduce the IgE-binding capacity of whey protein isolate (WPI) through glycation with dextran (DX). Blood sera were obtained from 8 patients who had been diagnosed with cow milk protein allergy, and a composite sera sample was used for IgE-binding analysis by the ImmunoCap (Phadia, Uppsala, Sweden) method. The WPI was glycated with DX of M W ranging from 1 to 2,000 kDa, and the M W of purified glycates was determined using size-exclusion chromatography coupled with multiangle laser light scattering. The WPI to DX molar ratios in the glycates made from DX that had M W values of 1, 3.5, 10 (G10), 150, 500, and 2,000 kDa were 1:4, 1:3, 1:2, 1:1.5, 1:1, and 1:1, respectively. With the increase in the M W of DX, there was an increase in the M W values of the corresponding glycates but a decrease in the number of bound DX. The WPI-DX glycates had lower whey protein IgE-binding capacity than native WPI, with the lowest IgE-binding capacity obtained in the G10 glycate. The DX binding ratios and morphology results from atomic force microscopy images suggested that glycation of WPI with small-M W DX resulted in extensive protein surface coverage, probably due to the attachment of up to 4 DX molecules per whey protein. The lower IgE binding of the G10 glycate was likely due to greater steric hindrance (or a physical barrier) at the surface of the protein. In summary, our results demonstrate that glycating WPI with DX via Maillard reaction can potentially be used to decrease the allergenicity of whey protein. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

PubMed Central

Hall Sedlak, Ruth; Hnilova, Marketa; Grosh, Carolynn; Fong, Hanson; Baneyx, Francois; Schwartz, Dan; Sarikaya, Mehmet; Tamerler, Candan

2012-01-01

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo. PMID:22286990

Nucleic acids encoding human trithorax protein

DOEpatents

Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

2001-01-01

In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

Fluorescence spectroscopy studies of HEK293 cells expressing DOR-Gi1α fusion protein; the effect of cholesterol depletion.

PubMed

Brejchová, Jana; Sýkora, Jan; Dlouhá, Kateřina; Roubalová, Lenka; Ostašov, Pavel; Vošahlíková, Miroslava; Hof, Martin; Svoboda, Petr

2011-12-01

Biophysical studies of fluorescence anisotropy of DPH and Laurdan generalized polarization were performed in plasma membranes (PM) isolated from control and cholesterol-depleted HEK293 cells stably expressing pertussis toxin (PTX)-insensitive DOR-Gi1α (Cys351-Ile351) fusion protein. PM isolated from control, PTX-untreated, cells were compared with PM isolated from PTX-treated cells. Results from both types of PM indicated that i) hydrophobic membrane interior was made more accessible to water molecules and more chaotically organized in cholesterol-depleted samples, ii) cholesterol depletion resulted in an overall increase in surface area of membrane, membrane fluidity, and mobility of its constituents. Analysis of DOR-Gi1α coupling in PTX-treated and PTX-untreated cells indicated that cholesterol depletion did not alter the agonist binding site of DOR (Bmax and Kd) but the ability of DOR agonist DADLE to activate G proteins was markedly impaired. In PTX-untreated membranes, EC50 for DADLE-stimulated [35S]GTPγS binding was shifted by one order of magnitude to the right: from 4.3±1.2×10(-9) M to 2.2±1.3×10(-8) M in control and cholesterol-depleted membrane samples, respectively. In PTX-treated membranes, EC50 was shifted from 4.5±1.1×10(-9) M to 2.8±1.4×10(-8) M. In summary, the perturbation of optimum PM organization by cholesterol depletion deteriorates functional coupling of DOR to covalently bound Gi1α as well as endogenously expressed PTX-sensitive G proteins of Gi/Go family while receptor ligand binding site is unchanged. The biophysical state of hydrophobic plasma (cell) membrane interior should be regarded as regulatory factor of DOR-signaling cascade. Copyright © 2011 Elsevier B.V. All rights reserved.

The crystal structure of a cyanobacterial water-soluble carotenoid binding protein.

PubMed

Kerfeld, Cheryl A; Sawaya, Michael R; Brahmandam, Vishnu; Cascio, Duilio; Ho, Kwok Ki; Trevithick-Sutton, Colleen C; Krogmann, David W; Yeates, Todd O

2003-01-01

Carotenoids undergo a wide range of photochemical reactions in animal, plant, and microbial systems. In photosynthetic organisms, in addition to light harvesting, they perform an essential role in protecting against light-induced damage by quenching singlet oxygen, superoxide anion radicals, or triplet-state chlorophyll. We have determined the crystal structure of a water-soluble orange carotenoid protein (OCP) isolated from the cyanobacterium Arthrospira maxima at a resolution of 2.1 A. OCP forms a homodimer with one carotenoid molecule per monomer. The carotenoid binding site is lined by a striking number of methionine residues. The structure reveals several possible ways in which the protein environment influences the spectral properties of the pigment and provides insight into how the OCP carries out its putative functions in photoprotection.

Expressing a bacterial mercuric ion binding protein in plant for phytoremediation of heavy metals.

PubMed

Hsieh, Ju-Liang; Chen, Ching-Yi; Chiu, Meng-Hsuen; Chein, Mei-Fang; Chang, Jo-Shu; Endo, Ginro; Huang, Chieh-Chen

2009-01-30

A specific mercuric ion binding protein (MerP) originating from transposon TnMERI1 of Bacillus megaterium strain MB1 isolated from Minamata Bay displayed good adsorption capability for a variety of heavy metals. In this study, the Gram-positive MerP protein was expressed in transgenic Arabidopsis to create a model system for phytoremediation of heavy metals. Under control of an actin promoter, the transgenic Arabidpsis showed higher tolerance and accumulation capacity for mercury, cadium and lead when compared with the control plant. Results from confocal microscopy analysis also indicate that MerP was localized at the cell membrane and vesicles of plant cells. The developed transgenic plants possessing excellent metal-accumulative ability could have potential applications in decontamination of heavy metals.

In situ dissection of RNA functional subunits by domain-specific chromatin isolation by RNA purification (dChIRP).

PubMed

Quinn, Jeffrey J; Chang, Howard Y

2015-01-01

Here we describe domain-specific chromatin isolation by RNA purification (dChIRP), a technique for dissecting the functional domains of a target RNA in situ. For an RNA of interest, dChIRP can identify domain-level intramolecular and intermolecular RNA-RNA, RNA-protein, and RNA-DNA interactions and maps the RNA's genomic binding sites with higher precision than domain-agnostic methods. We illustrate how this technique has been applied to the roX1 lncRNA to resolve its domain-level architecture, discover its protein- and chromatin-interacting domains, and map its occupancy on the X chromosome.

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

Wu, R.; Wilton, R.; Cuff, M. E.

The tandem Per-Arnt-Sim (PAS) like sensors are commonly found in signal transduction proteins. The periplasmic solute binding protein (SBP) domains are found ubiquitously and are generally involved in solute transport. These domains are widely observed as parts of separate proteins but not within the same polypeptide chain. We report the structural and biochemical characterization of the extracellular ligand-binding receptor, Dret_0059 from Desulfohalobium retbaense DSM 5692, an organism isolated from the Retba salt lake in Senegal. The structure of Dret_0059 consists of a novel combination of SBP and TPAS sensor domains. The N-terminal region forms an SBP domain and the C-terminalmore » region folds into a tandem PAS-like domain structure. A ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS domain of the TPAS. The differential scanning flourimetry studies in solution support the ligands observed in the crystal structure. There are only two other proteins with this structural architecture in the non-redundant sequence data base and we predict that they too bind the same substrates. There is significant interaction between the SBP and TPAS domains, and it is quite conceivable that the binding of one ligand will have an effect on the binding of the other. Our attempts to remove the ligands bound to the protein during expression were not successful, therefore, it is not clear what the relative affects are. The genomic context of this receptor does not contain any protein components expected for transport function, hence, we suggest that Dret_0059 is likely involved in signal transduction and not in solute transport.« less

Glutamate receptors as seen by light: Spectroscopic studies of structure-function relationships

PubMed Central

Mankiewicz, Kimberly A.; Jayaraman, Vasanthi

2010-01-01

Ionotropic glutamate receptors are major excitatory receptors in the central nervous system and also have far reaching influence in other areas of the body. Their modular nature has allowed for the isolation of the ligand binding domain and subsequent structural studies using a variety of spectroscopic techniques. This review will discuss the role of specific ligand:protein interactions in mediating activation in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptors as established by various spectroscopic investigations of the GluR2 and GluR4 subunits of this receptor. Specifically, this review will provide an introduction of the insight gained from X-ray crystallography and nuclear magnetic resonance (NMR) investigations and then go on to focus on studies utilizing vibrational spectroscopy and fluorescence resonance energy transfer (FRET) to study the behavior of the isolated ligand binding domain in solution and discuss the importance of specific ligand:protein interactions in the mechanism of receptor activation. PMID:17934637

The Role of IQGAP1 in Breast Carcinoma

DTIC Science & Technology

2012-10-01

and"-tubulin expression was measured as described above. Statistical Analysis —All experiments were repeated inde- pendently at least three times...IQGAP1 Binds HER2—In vitro analysis with pure proteins was used to examine a possible interaction between IQGAP1 and HER2. GST alone or GST-HER2 was...incubated with puri- fied IQGAP1, and complexes were isolated with glutathione- Sepharose. Analysis by Western blotting reveals that IQGAP1 bindsHER2

A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity.

PubMed

Lindberg, Hanna; Härd, Torleif; Löfblom, John; Ståhl, Stefan

2015-09-01

The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide. © 2015 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-Non-Commercial-NoDerivs Licence, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Human Brain-Derived Aβ Oligomers Bind to Synapses and Disrupt Synaptic Activity in a Manner That Requires APP.

PubMed

Wang, Zemin; Jackson, Rosemary J; Hong, Wei; Taylor, Walter M; Corbett, Grant T; Moreno, Arturo; Liu, Wen; Li, Shaomin; Frosch, Matthew P; Slutsky, Inna; Young-Pearse, Tracy L; Spires-Jones, Tara L; Walsh, Dominic M

2017-12-06

Compelling genetic evidence links the amyloid precursor protein (APP) to Alzheimer's disease (AD) and several theories have been advanced to explain the relationship. A leading hypothesis proposes that a small amphipathic fragment of APP, the amyloid β-protein (Aβ), self-associates to form soluble aggregates that impair synaptic and network activity. Here, we used the most disease-relevant form of Aβ, protein isolated from AD brain. Using this material, we show that the synaptotoxic effects of Aβ depend on expression of APP and that the Aβ-mediated impairment of synaptic plasticity is accompanied by presynaptic effects that disrupt the excitatory/inhibitory (E/I) balance. The net increase in the E/I ratio and inhibition of plasticity are associated with Aβ localizing to synapses and binding of soluble Aβ aggregates to synapses requires the expression of APP. Our findings indicate a role for APP in AD pathogenesis beyond the generation of Aβ and suggest modulation of APP expression as a therapy for AD. SIGNIFICANCE STATEMENT Here, we report on the plasticity-disrupting effects of amyloid β-protein (Aβ) isolated from Alzheimer's disease (AD) brain and the requirement of amyloid precursor protein (APP) for these effects. We show that Aβ-containing AD brain extracts block hippocampal LTP, augment glutamate release probability, and disrupt the excitatory/inhibitory balance. These effects are associated with Aβ localizing to synapses and genetic ablation of APP prevents both Aβ binding and Aβ-mediated synaptic dysfunctions. Our results emphasize the importance of APP in AD and should stimulate new studies to elucidate APP-related targets suitable for pharmacological manipulation. Copyright © 2017 the authors 0270-6474/17/3711947-20$15.00/0.

Development of a Novel Human scFv Against EGFR L2 Domain by Phage Display Technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Khosroshahi, Shiva Ahdi; Tanomand, Asghar

2017-01-01

Epidermal growth factor receptor (EGFR) as a transmembrane tyrosine kinase receptor frequently overexpresses in tumors with epithelial origin. The L2 domain from extracellular part of EGFR is involved in ligand binding and the blockage of this domain prevents activation of related signaling pathways. This study was aimed to develop a novel human scFv against EGFR L2 domain as a promising target for cancer therapy. The L2 recombinant protein was purified and used for panning a human scFv phage library (Tomlinson I). In this study, a novel screening strategy was applied to select clones with high binding and enrichment of rare specific phage clones of the L2 protein. After five biopanning rounds several specific clones were isolated which among them one phage clone with high binding was purified for further analysis. The specific interaction of selected clone against target antigen was confirmed by ELISA and western blotting. Immunofluorescence staining showed that purified scFv binds to A431 cells surface, displaying EGFR surface receptor. In the present study, we isolated for the first time a novel human scFv against EGFR L2 domain. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFR overexpressing cancers using this novel human anti-L2 ScFv. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein

NASA Technical Reports Server (NTRS)

Salins, L. L.; Ware, R. A.; Ensor, C. M.; Daunert, S.

2001-01-01

The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible. Copyright 2001 Academic Press.

Iron Uptake Mechanisms in the Fish Pathogen Tenacibaculum maritimum

PubMed Central

Avendaño-Herrera, Ruben; Toranzo, Alicia E.; Romalde, Jesús L.; Lemos, Manuel L.; Magariños, Beatriz

2005-01-01

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen Tenacibaculum maritimum. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di- (o-hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all T. maritimum strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all T. maritimum strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the T. maritimum cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that T. maritimum possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding. PMID:16269729

Iron uptake mechanisms in the fish pathogen Tenacibaculum maritimum.

PubMed

Avendaño-Herrera, Ruben; Toranzo, Alicia E; Romalde, Jesús L; Lemos, Manuel L; Magariños, Beatriz

2005-11-01

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen Tenacibaculum maritimum. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di-(o-hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all T. maritimum strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all T. maritimum strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the T. maritimum cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. Proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that T. maritimum possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding.

Formation of difluorothionoacetyl-protein adducts by S-(1,1,2,2-tetrafluoroethyl)-L-cysteine metabolites: Nucleophilic catalysis of stable lysyl adduct formation by histidine and tyrosine

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

Hayden, P.J.; McCann, D.J.; Stevens, J.L.

1991-06-18

{sup 19}F NMR spectorscopy was used in conjunction with isotopic labeling to demonstrate that difluorothionoacetyl-protein adducts are formed by metabolites of the nephrotoxic cysteine conjugate S(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC). To determine which amino acid residues can be involved in adduct formation, the reactivity of TFEC metabolites with a variety of N-acetyl amino acids was also investigated. An N{sup {alpha}}-acetyl-N{sup {epsilon}}-(difluorothionoacetyl)lysine (DFTAL) adduct was isolated and characterized by {sup 19}F and {sup 13}C NMR spectroscopy and mass spectrometry. N{sup {alpha}}-Acetylhistidine and N-acetyltyrosine were found to act as nucleophilic catalysts to facilitate the formation of both the protein and DFTAL adducts. Adduct formation wasmore » greatly reduced when lysyl-modified protein was used as the substrate, indicating that lysyl residues are primary sites of adduct formation. However, N{sup a}-acetyllysine, at concentrations of >100-fold in excess compared to protein lysyl residues, was not effective in preventing binding of metabolites to protein. Therefore, nucleophilic catalysis at the surface of the protein may be an important mechanism for the binding of TFEC metabolites to specific lysyl residues in protein. TFEC metabolites were very reactive with the thiol nucleophiles glutathione and N-acetylcysteine. However, the predicted difluorodithioesters could not be isolated. Bothe stable difluorothioacetamide and less stable difluorodithioester protein adducts may play a role in TFEC-mediated enphrotoxicity.« less

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm.

PubMed

Coux, Gabriela; Cabada, Marcelo O

2006-04-28

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.

Interactions between peroxiredoxin 2, hemichrome and the erythrocyte membrane.

PubMed

Bayer, Simone B; Low, Felicia M; Hampton, Mark B; Winterbourn, Christine C

2016-12-01

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H 2 O 2 , but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H 2 O 2 or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.

Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles

PubMed Central

Labonté, Eric D.; Howles, Philip N.; Granholm, Norman A.; Rojas, Juan C.; Davies, Joanna P.; Ioannou, Yiannis A.; Hui, David Y.

2007-01-01

Recent studies have documented the importance of Niemann Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1−/− mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1−/− mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1−/− mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1. PMID:17442616

Relationship of calcium and membrane guanylate cyclase in adrenocorticotropin-induced steroidogenesis.

PubMed

Nambi, P; Aiyar, N V; Roberts, A N; Sharma, R K

1982-07-01

Chlorpromazine, when incubated with isolated adrenal cells, inhibited the ACTH-stimulated formation of cGMP and corticosterone production. It also inhibited the ACTH-stimulated membrane guanylate cyclase, but did not affect the binding of ACTH to the membrane receptors. cGMP-induced steroidogenesis was not affected by the drug. These data indicate that chlorpromazine interferes with adrenal steroid metabolism at a site between the hormone receptor and guanylate cyclase and also show that guanylate cyclase is composed of separate receptor and catalytic components. Furthermore, based on the premise that chlorpromazine exerts its inhibitory action by blocking the binding of a calcium receptor protein, such as calmodulin, to the receptor-coupled guanylate cyclase, it is proposed that the interaction of calcium, presumably through a calcium-binding protein, is essential for ACTH-dependent guanylate cyclase.

Recent progress in elucidating the structure, function and evolution of disease resistance genes in plants.

PubMed

Liu, Jinling; Liu, Xionglun; Dai, Liangying; Wang, Guoliang

2007-09-01

Plants employ multifaceted mechanisms to fight with numerous pathogens in nature. Resistance (R) genes are the most effective weapons against pathogen invasion since they can specifically recognize the corresponding pathogen effectors or associated protein(s) to activate plant immune responses at the site of infection. Up to date, over 70 R genes have been isolated from various plant species. Most R proteins contain conserved motifs such as nucleotide-binding site (NBS), leucine-rich repeat (LRR), Toll-interleukin-1 receptor domain (TIR, homologous to cytoplasmic domains of the Drosophila Toll protein and the mammalian interleukin-1 receptor), coiled-coil (CC) or leucine zipper (LZ) structure and protein kinase domain (PK). Recent results indicate that these domains play significant roles in R protein interactions with effector proteins from pathogens and in activating signal transduction pathways involved in innate immunity. This review highlights an overview of the recent progress in elucidating the structure, function and evolution of the isolated R genes in different plant-pathogen interaction systems.

Lignin Films from Spruce, Eucalyptus, and Wheat Straw Studied with Electroacoustic and Optical Sensors: Effect of Composition and Electrostatic Screening on Enzyme Binding.

PubMed

Pereira, Antonio; Hoeger, Ingrid C; Ferrer, Ana; Rencoret, Jorge; Del Rio, José C; Kruus, Kristiina; Rahikainen, Jenni; Kellock, Miriam; Gutiérrez, Ana; Rojas, Orlando J

2017-04-10

Lignins were isolated from spruce, wheat straw, and eucalyptus by using the milled wood lignin (MWL) method. Functional groups and compositional analyses were assessed via 2D NMR and 31 P NMR to realize their effect on enzyme binding. Films of the lignins were fabricated and ellipsometry, atomic force microscopy, and water contact angle measurements were used for their characterization and to reveal the changes upon enzyme adsorption. Moreover, lignin thin films were deposited on quartz crystal microgravimetry (QCM) and surface plasmon (SPR) resonance sensors and used to gain further insights into the lignin-cellulase interactions. For this purpose, a commercial multicomponent enzyme system and a monocomponent Trichoderma reesei exoglucanase (CBH-I) were considered. Strong enzyme adsorption was observed on the various lignins but compared to the multicomponent cellulases, CBH-I displayed lower surface affinity and higher binding reversibility. This resolved prevalent questions related to the affinity of this enzyme with lignin. Remarkably, a strong correlation between enzyme binding and the syringyl/guaiacyl (S/G) ratio was found for the lignins, which presented a similar hydroxyl group content ( 31 P NMR): higher protein affinity was determined on isolated spruce lignin (99% G units), while the lowest adsorption occurred on isolated eucalyptus lignin (70% S units). The effect of electrostatic interactions in enzyme adsorption was investigated by SPR, which clearly indicated that the screening of charges allowed more extensive protein adsorption. Overall, this work furthers our understanding of lignin-cellulase interactions relevant to biomass that has been subjected to no or little pretreatment and highlights the widely contrasting effects of the nature of lignin, which gives guidance to improve lignocellulosic saccharification and related processes.

Molecular cloning and evolutionary analysis of the calcium-modulated contractile protein, centrin, in green algae and land plants.

PubMed

Bhattacharya, D; Steinkötter, J; Melkonian, M

1993-12-01

Centrin (= caltractin) is a ubiquitous, cytoskeletal protein which is a member of the EF-hand superfamily of calcium-binding proteins. A centrin-coding cDNA was isolated and characterized from the prasinophyte green alga Scherffelia dubia. Centrin PCR amplification primers were used to isolate partial, homologous cDNA sequences from the green algae Tetraselmis striata and Spermatozopsis similis. Annealing analyses suggested that centrin is a single-copy-coding region in T. striata and S. similis and other green algae studied. Centrin-coding regions from S. dubia, S. similis and T. striata encode four colinear EF-hand domains which putatively bind calcium. Phylogenetic analyses, including homologous sequences from Chlamydomonas reinhardtii and the land plant Atriplex nummularia, demonstrate that the domains of centrins are congruent and arose from the two-fold duplication of an ancestral EF hand with Domains 1+3 and Domains 2+4 clustering. The domains of centrins are also congruent with those of calmodulins demonstrating that, like calmodulin, centrin is an ancient protein which arose within the ancestor of all eukaryotes via gene duplication. Phylogenetic relationships inferred from centrin-coding region comparisons mirror results of small subunit ribosomal RNA sequence analyses suggesting that centrin-coding regions are useful evolutionary markers within the green algae.

Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition.

PubMed

Neth, O; Jack, D L; Dodds, A W; Holzel, H; Klein, N J; Turner, M W

2000-02-01

Mannose-binding lectin (MBL) is a collagenous serum lectin believed to be of importance in innate immunity. Genetically determined low levels of the protein are known to predispose to infections. In this study the binding of purified MBL to pathogens isolated from immunocompromised children was investigated by flow cytometry. Diverse Candida species, Aspergillus fumigatus, Staphylococcus aureus, and beta-hemolytic group A streptococci exhibited strong binding of MBL, whereas Escherichia coli, Klebsiella species, and Haemophilus influenzae type b were characterized by heterogeneous binding patterns. In contrast, beta-hemolytic group B streptococci, Streptococcus pneumoniae, and Staphylococcus epidermidis showed low levels of binding. Bound MBL was able to promote C4 deposition in a concentration-dependent manner. We conclude that MBL may be of importance in first-line immune defense against several important pathogens.

The genomic structure of the human Charcot-Leyden crystal protein gene is analogous to those of the galectin genes

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

Dyer, K.D.; Handen, J.S.; Rosenberg, H.F.

The Charcot-Leyden crystal (CLC) protein, or eosinophil lysophospholipase, is a characteristic protein of human eosinophils and basophils; recent work has demonstrated that the CLC protein is both structurally and functionally related to the galectin family of {beta}-galactoside binding proteins. The galectins as a group share a number of features in common, including a linear ligand binding site encoded on a single exon. In this work, we demonstrate that the intron-exon structure of the gene encoding CLC is analogous to those encoding the galectins. The coding sequence of the CLC gene is divided into four exons, with the entire {beta}-galactoside bindingmore » site encoded by exon III. We have isolated CLC {beta}-galactoside binding sites from both orangutan (Pongo pygmaeus) and murine (Mus musculus) genomic DNAs, both encoded on single exons, and noted conservation of the amino acids shown to interact directly with the {beta}-galactoside ligand. The most likely interpretation of these results suggests the occurrence of one or more exon duplication and insertion events, resulting in the distribution of this lectin domain to CLC as well as to the multiple galectin genes. 35 refs., 3 figs.« less

Structural study of the Fox-1 RRM protein hydration reveals a role for key water molecules in RRM-RNA recognition

PubMed Central

Blatter, Markus; Cléry, Antoine; Damberger, Fred F.

2017-01-01

Abstract The Fox-1 RNA recognition motif (RRM) domain is an important member of the RRM protein family. We report a 1.8 Å X-ray structure of the free Fox-1 containing six distinct monomers. We use this and the nuclear magnetic resonance (NMR) structure of the Fox-1 protein/RNA complex for molecular dynamics (MD) analyses of the structured hydration. The individual monomers of the X-ray structure show diverse hydration patterns, however, MD excellently reproduces the most occupied hydration sites. Simulations of the protein/RNA complex show hydration consistent with the isolated protein complemented by hydration sites specific to the protein/RNA interface. MD predicts intricate hydration sites with water-binding times extending up to hundreds of nanoseconds. We characterize two of them using NMR spectroscopy, RNA binding with switchSENSE and free-energy calculations of mutant proteins. Both hydration sites are experimentally confirmed and their abolishment reduces the binding free-energy. A quantitative agreement between theory and experiment is achieved for the S155A substitution but not for the S122A mutant. The S155 hydration site is evolutionarily conserved within the RRM domains. In conclusion, MD is an effective tool for predicting and interpreting the hydration patterns of protein/RNA complexes. Hydration is not easily detectable in NMR experiments but can affect stability of protein/RNA complexes. PMID:28505313

An Analysis of the Binding Characteristics of a Panel of Recently Selected ICAM-1 Binding Plasmodium falciparum Patient Isolates

PubMed Central

Madkhali, Aymen M.; Alkurbi, Mohammed O.; Szestak, Tadge; Bengtsson, Anja; Patil, Pradeep R.; Wu, Yang; Alharthi, Saeed; Jensen, Anja T. R.; Pleass, Richard; Craig, Alister G.

2014-01-01

The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE) from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1) expressed on the surface of the infected erythrocyte membrane. One of the commonly used host receptors is ICAM-1, and it has been suggested that ICAM-1 has a role in cerebral malaria pathology, although the evidence to support this is not conclusive. The current study examined the cytoadherence patterns of lab-adapted patient isolates after selecting on ICAM-1. We investigated the binding phenotypes using variant ICAM-1 proteins including ICAM-1Ref, ICAM-1Kilifi, ICAM-1S22/A, ICAM-1L42/A and ICAM-1L44/A using static assays. The study also examined ICAM-1 blocking by four anti-ICAM-1 monoclonal antibodies (mAb) under static conditions. We also characterised the binding phenotypes using Human Dermal Microvascular Endothelial Cells (HDMEC) under flow conditions. The results show that different isolates have variant-specific binding phenotypes under both static and flow conditions, extending our previous observations that this variation might be due to variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants. PMID:25360558

Acquisition of haemoglobin-bound iron by strains of the Actinobacillus minor/"porcitonsillarum" complex.

PubMed

Arya, Gitanjali; Niven, Donald F

2011-03-24

Members of the Actinobacillus minor/"porcitonsillarum" complex are common inhabitants of the swine respiratory tract. Although avirulent or of low virulence for pigs, these organisms, like pathogens, do grow in vivo and must, therefore, be able to acquire iron within the host. Here, we investigated the abilities of six members of the A. minor/"porcitonsillarum" complex to acquire iron from transferrin and various haemoglobins. Using growth assays, all six strains were shown to acquire iron from porcine, bovine and human haemoglobins but not from porcine transferrin. Analyses of whole genome sequences revealed that A. minor strains NM305(T) and 202, unlike the swine-pathogenic actinobacilli, A. pleuropneumoniae and A. suis, lack not only the transferrin-binding protein genes, tbpA and tbpB, but also the haemoglobin-binding protein gene, hgbA. Strains NM305(T) and 202, however, were found to possess other putative haemin/haemoglobin-binding protein genes that were predicted to encode mature proteins of ∼ 72 and ∼ 75 kDa, respectively. An affinity procedure based on haemin-agarose allowed the isolation of ∼ 65 and ∼ 67 kDa iron-repressible outer membrane polypeptides from membranes derived from strains NM305(T) and 202, respectively, and mass spectrometry revealed that these polypeptides were the products of the putative haemin/haemoglobin-binding protein genes. PCR approaches allowed the amplification and sequencing of homologues of both haemin/haemoglobin-binding protein genes from each of the other four strains, strains 33PN and 7ATS of the A. minor/"porcitonsillarum" complex and "A. porcitonsillarum" strains 9953L55 and 0347, suggesting that such proteins are involved in the utilization of haemoglobin-bound iron, presumably as surface receptors, by all six strains investigated. Copyright © 2010 Elsevier B.V. All rights reserved.

Mammalian Peptidylglycine α-Amidating Monooxygenase mRNA Expression Can Be Modulated by the La Autoantigen

PubMed Central

Brenet, Fabienne; Dussault, Nadège; Borch, Jonas; Ferracci, Géraldine; Delfino, Christine; Roepstorff, Peter; Miquelis, Raymond; Ouafik, L'Houcine

2005-01-01

Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal α-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3′ untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3′ UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3′ UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3′ UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules. PMID:16107699

Organization, chromosomal localization and promoter analysis of the gene encoding human acidic fibroblast growth factor intracellular binding protein.

PubMed Central

Kolpakova, E; Frengen, E; Stokke, T; Olsnes, S

2000-01-01

Acidic fibroblast growth factor (aFGF) intracellular binding protein (FIBP) is a protein found mainly in the nucleus that might be involved in the intracellular function of aFGF. Here we present a comparative analysis of the deduced amino acid sequences of human, murine and Drosophila FIBP analogues and demonstrate that FIBP is an evolutionarily conserved protein. The human gene spans more than 5 kb, comprising ten exons and nine introns, and maps to chromosome 11q13.1. Two slightly different splice variants found in different tissues were isolated and characterized. Sequence analysis of the region surrounding the translation start revealed a CpG island, a classical feature of widely expressed genes. Functional studies of the promoter region with a luciferase reporter system suggested a strong transcriptional activity residing within 600 bp of the 5' flanking region. PMID:11104667

The C8-binding protein of human erythrocytes: interaction with the components of the complement-attack phase.

PubMed Central

Schönermark, S; Filsinger, S; Berger, B; Hänsch, G M

1988-01-01

C8-binding protein is an intrinsic membrane protein of the human erythrocyte. It inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. In the present study we incorporated C8bp, isolated from human erythrocytes, into sheep erythrocytes (SRBC). SRBC, normally sensitive to lysis by human C5b-9, became insensitive to lysis. Furthermore, we found that C8bp is incorporated into the membrane-attack complex C5b-9, most probably by interacting with C8, since C8bp has an affinity for C8, particularly for the C8 alpha-gamma-subunit. Antibodies to C8bp react with the C8 alpha-subunits and with C9, pointing to the possibility of a partial homology between these proteins. Images Figure 4 Figure 6 Figure 7 PMID:3366469

Purification of FKBP-70, a novel immunophilin from Saccharomyces cerevisiae, and cloning of its structural gene, FPR3.

PubMed

Manning-Krieg, U C; Henríquez, R; Cammas, F; Graff, P; Gavériaux, S; Movva, N R

1994-09-19

A novel protein, belonging to the yeast family of FKBPs (FK-binding proteins), FKBP-70, was isolated from Saccharomyces cerevisiae by its interaction with the immunosuppressive agent FK-520. Its structural gene, FPR3, was cloned and the protein expressed and purified from Escherichia coli. This third member of the FKBP family in yeast is homologous to the other FKBPs at its carboxy terminus, showing conserved ligand binding and proline isomerase regions. It is, however, a longer acidic protein with several potential nuclear targeting sequences and a region of homology to nucleolins. Yeast strains deleted for FPR3, as well as a triple deletion mutant of this family of genes, FPR1, FPR2 and FPR3, are viable under normal conditions of growth, indicating that the FPR genes are not essential for life.

Molecular cloning, expression and molecular modeling of chemosensory protein from Spodoptera litura and its binding properties with Rhodojaponin III.

PubMed

Zhang, Yanbo; Dong, Xiaolin; Liu, Jinxiang; Hu, Meiying; Zhong, Guohua; Geng, Peng; Yi, Xin

2012-01-01

Insects stimulate specific behaviors by the correct recognition of the chemicals in the external environment. Rhodojaponin III is a botanical grayanoid diterpenid oviposition deterrent isolated from Rhododendron molle. In this study we aimed to determine whether the CSPs involved in the recognition of Rhodojaponin III. A full-length cDNA encoding chemosensory protein was isolated from the antennae of Spodoptera litura Fabricius (CSPSlit, GenBank Accession No. DQ007458). The full-length cDNA of NlFoxA is 1789 bp and has an open reading frame (ORF) of 473 bp, encoding a protein of 126 amino acids, Northern blot analysis revealed that CSPSlit mRNA was mainly expressed in the antennae, legs, wings and female abdomens. A three-dimensional model of CSPSlit was constructed using homology modeling method, and its reliability was evaluated. The active site of CSPSlit was calculated using CDOCKER program indicated that the Tyr24, Ile45, Leu49, Thr64, Leu68, Trp79 and Leu82 were responsible ligand-binding active site on identifying Rhodojaponin III in the CSPSlit. The recombinant CSPSlit protein was expressed in Escherichia coli and purified using single-step Ni-NTA affinity chromatography. Fluorescence emission spectra revealed that the CSPSlit protein had significant affinity to rhodojaponin III. These results mean that CSPSlit is critical for insects identify the Rhodojaponin III.

Molecular Cloning, Expression and Molecular Modeling of Chemosensory Protein from Spodoptera litura and Its Binding Properties with Rhodojaponin III

PubMed Central

Zhang, Yanbo; Dong, Xiaolin; Liu, Jinxiang; Hu, Meiying; Zhong, Guohua; Geng, Peng; Yi, Xin

2012-01-01

Insects stimulate specific behaviors by the correct recognition of the chemicals in the external environment. Rhodojaponin III is a botanical grayanoid diterpenid oviposition deterrent isolated from Rhododendron molle. In this study we aimed to determine whether the CSPs involved in the recognition of Rhodojaponin III. A full-length cDNA encoding chemosensory protein was isolated from the antennae of Spodoptera litura Fabricius (CSPSlit, GenBank Accession No. DQ007458). The full-length cDNA of NlFoxA is 1789 bp and has an open reading frame (ORF) of 473 bp, encoding a protein of 126 amino acids, Northern blot analysis revealed that CSPSlit mRNA was mainly expressed in the antennae, legs, wings and female abdomens. A three-dimensional model of CSPSlit was constructed using homology modeling method, and its reliability was evaluated. The active site of CSPSlit was calculated using CDOCKER program indicated that the Tyr24, Ile45, Leu49, Thr64, Leu68, Trp79 and Leu82 were responsible ligand-binding active site on identifying Rhodojaponin III in the CSPSlit. The recombinant CSPSlit protein was expressed in Escherichia coli and purified using single-step Ni-NTA affinity chromatography. Fluorescence emission spectra revealed that the CSPSlit protein had significant affinity to rhodojaponin III. These results mean that CSPSlit is critical for insects identify the Rhodojaponin III. PMID:23133516

Diversity and cold adaptation of microorganisms isolated from the Schirmacher Oasis, Antarctica

NASA Astrophysics Data System (ADS)

Mojib, Nazia; Bej, Asim K.; Hoover, Richard

2008-08-01

We have investigated the feasibility of the PCR amplification of the 16S rRNA genes from eubacteria and Archea on samples collected on Whatman FTA filters from Schirmacher Oasis for the study of culture-independent analysis of the microbial diversity. Both conventional PCR and real-time TaqmaTM PCR successfully amplified the targeted genes. A number of diverse groups of psychrotolerant microorganisms with various pigments have been isolated when cultured on agar medium. 16S rRNA gene analysis of these isolates helped us to identify closest taxonomic genus Pseudomonas, Frigoribacterium, Arthrobacter, Flavobacterium, and Janthinobacterium. It is possible that the pigments play protective role from solar UV radiation, which is prevalent in Antarctic continent especially during Austral summer months. Study of the expression of cold adaptive protein CapB and ice-binding protein IBP using western blots showed positive detection of both or either of these proteins in 6 out of 8 isolates. Since the CapB and IBP protein structure greatly varies in microorganisms, it is possible that the 2 isolates with negative results could have a different class of these proteins. The expression of the CapB and the IBP in these isolates suggest that these proteins are essential for the survival in the Antarctic cold and subzero temperatures and protect themselves from freeze-damage. The current study provided sufficient data to further investigate the rich and diverse biota of psychrotolerant extremophiles in the Antarctic Schirmacher Oasis using both culture-independent and culture-based approaches; and understand the mechanisms of cold tolerance.

Sarcomeric protein modification during adrenergic stress enhances cross-bridge kinetics and cardiac output

PubMed Central

Gresham, Kenneth S.; Mamidi, Ranganath; Li, Jiayang; Kwak, Hyerin

2017-01-01

Molecular adaptations to chronic neurohormonal stress, including sarcomeric protein cleavage and phosphorylation, provide a mechanism to increase ventricular contractility and enhance cardiac output, yet the link between sarcomeric protein modifications and changes in myocardial function remains unclear. To examine the effects of neurohormonal stress on posttranslational modifications of sarcomeric proteins, mice were administered combined α- and β-adrenergic receptor agonists (isoproterenol and phenylephrine, IPE) for 14 days using implantable osmotic pumps. In addition to significant cardiac hypertrophy and increased maximal ventricular pressure, IPE treatment accelerated pressure development and relaxation (74% increase in dP/dtmax and 14% decrease in τ), resulting in a 52% increase in cardiac output compared with saline (SAL)-treated mice. Accelerated pressure development was maintained when accounting for changes in heart rate and preload, suggesting that myocardial adaptations contribute to enhanced ventricular contractility. Ventricular myocardium isolated from IPE-treated mice displayed a significant reduction in troponin I (TnI) and myosin-binding protein C (MyBP-C) expression and a concomitant increase in the phosphorylation levels of the remaining TnI and MyBP-C protein compared with myocardium isolated from saline-treated control mice. Skinned myocardium isolated from IPE-treated mice displayed a significant acceleration in the rate of cross-bridge (XB) detachment (46% increase) and an enhanced magnitude of XB recruitment (43% increase) at submaximal Ca2+ activation compared with SAL-treated mice but unaltered myofilament Ca2+ sensitivity of force generation. These findings demonstrate that sarcomeric protein modifications during neurohormonal stress are molecular adaptations that enhance in vivo ventricular contractility through accelerated XB kinetics to increase cardiac output. NEW & NOTEWORTHY Posttranslational modifications to sarcomeric regulatory proteins provide a mechanism to modulate cardiac function in response to stress. In this study, we demonstrate that neurohormonal stress produces modifications to myosin-binding protein C and troponin I, including a reduction in protein expression within the sarcomere and increased phosphorylation of the remaining protein, which serve to enhance cross-bridge kinetics and increase cardiac output. These findings highlight the importance of sarcomeric regulatory protein modifications in modulating ventricular function during cardiac stress. PMID:27909224

Sarcomeric protein modification during adrenergic stress enhances cross-bridge kinetics and cardiac output.

PubMed

Gresham, Kenneth S; Mamidi, Ranganath; Li, Jiayang; Kwak, Hyerin; Stelzer, Julian E

2017-03-01

Molecular adaptations to chronic neurohormonal stress, including sarcomeric protein cleavage and phosphorylation, provide a mechanism to increase ventricular contractility and enhance cardiac output, yet the link between sarcomeric protein modifications and changes in myocardial function remains unclear. To examine the effects of neurohormonal stress on posttranslational modifications of sarcomeric proteins, mice were administered combined α- and β-adrenergic receptor agonists (isoproterenol and phenylephrine, IPE) for 14 days using implantable osmotic pumps. In addition to significant cardiac hypertrophy and increased maximal ventricular pressure, IPE treatment accelerated pressure development and relaxation (74% increase in dP/d t max and 14% decrease in τ), resulting in a 52% increase in cardiac output compared with saline (SAL)-treated mice. Accelerated pressure development was maintained when accounting for changes in heart rate and preload, suggesting that myocardial adaptations contribute to enhanced ventricular contractility. Ventricular myocardium isolated from IPE-treated mice displayed a significant reduction in troponin I (TnI) and myosin-binding protein C (MyBP-C) expression and a concomitant increase in the phosphorylation levels of the remaining TnI and MyBP-C protein compared with myocardium isolated from saline-treated control mice. Skinned myocardium isolated from IPE-treated mice displayed a significant acceleration in the rate of cross-bridge (XB) detachment (46% increase) and an enhanced magnitude of XB recruitment (43% increase) at submaximal Ca 2+ activation compared with SAL-treated mice but unaltered myofilament Ca 2+ sensitivity of force generation. These findings demonstrate that sarcomeric protein modifications during neurohormonal stress are molecular adaptations that enhance in vivo ventricular contractility through accelerated XB kinetics to increase cardiac output. NEW & NOTEWORTHY Posttranslational modifications to sarcomeric regulatory proteins provide a mechanism to modulate cardiac function in response to stress. In this study, we demonstrate that neurohormonal stress produces modifications to myosin-binding protein C and troponin I, including a reduction in protein expression within the sarcomere and increased phosphorylation of the remaining protein, which serve to enhance cross-bridge kinetics and increase cardiac output. These findings highlight the importance of sarcomeric regulatory protein modifications in modulating ventricular function during cardiac stress. Copyright © 2017 the American Physiological Society.

ABFs, a family of ABA-responsive element binding factors.

PubMed

Choi, H; Hong, J; Ha, J; Kang, J; Kim, S Y

2000-01-21

Abscisic acid (ABA) plays an important role in environmental stress responses of higher plants during vegetative growth. One of the ABA-mediated responses is the induced expression of a large number of genes, which is mediated by cis-regulatory elements known as abscisic acid-responsive elements (ABREs). Although a number of ABRE binding transcription factors have been known, they are not specifically from vegetative tissues under induced conditions. Considering the tissue specificity of ABA signaling pathways, factors mediating ABA-dependent stress responses during vegetative growth phase may thus have been unidentified so far. Here, we report a family of ABRE binding factors isolated from young Arabidopsis plants under stress conditions. The factors, isolated by a yeast one-hybrid system using a prototypical ABRE and named as ABFs (ABRE binding factors) belong to a distinct subfamily of bZIP proteins. Binding site selection assay performed with one ABF showed that its preferred binding site is the strong ABRE, CACGTGGC. ABFs can transactivate an ABRE-containing reporter gene in yeast. Expression of ABFs is induced by ABA and various stress treatments, whereas their induction patterns are different from one another. Thus, a new family of ABRE binding factors indeed exists that have the potential to activate a large number of ABA/stress-responsive genes in Arabidopsis.

Fibrinogen-binding and platelet-aggregation activities of a Lactobacillus salivarius septicaemia isolate are mediated by a novel fibrinogen-binding protein.

PubMed

Collins, James; van Pijkeren, Jan-Peter; Svensson, Lisbeth; Claesson, Marcus J; Sturme, Mark; Li, Yin; Cooney, Jakki C; van Sinderen, Douwe; Walker, Alan W; Parkhill, Julian; Shannon, Oonagh; O'Toole, Paul W

2012-09-01

The marketplace for probiotic foods is burgeoning, measured in billions of euro per annum. It is imperative, however, that all bacterial strains are fully assessed for human safety. The ability to bind fibrinogen is considered a potential pathogenicity trait that can lead to platelet aggregation, serious medical complications, and in some instances, death. Here we examined strains from species frequently used as probiotics for their ability to bind human fibrinogen. Only one strain (CCUG 47825), a Lactobacillus salivarius isolate from a case of septicaemia, was found to strongly adhere to fibrinogen. Furthermore, this strain was found to aggregate human platelets at a level comparable to the human pathogen Staphylococcus aureus. By sequencing the genome of CCUG 47825, we were able to identify candidate genes responsible for fibrinogen binding. Complementing the genetic analysis with traditional molecular microbiological techniques enabled the identification of the novel fibrinogen receptor, CCUG_2371. Although only strain CCUG 47825 bound fibrinogen under laboratory conditions, homologues of the novel fibrinogen binding gene CCUG_2371 are widespread among L. salivarius strains, maintaining their potential to bind fibrinogen if expressed. We highlight the fact that without a full genetic analysis of strains for human consumption, potential pathogenicity traits may go undetected. © 2012 Blackwell Publishing Ltd.

Solid-phase glycan isolation for glycomics analysis

PubMed Central

Yang, Shuang; Zhang, Hui

2013-01-01

Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for glycomics analysis. PMID:23090885

Solid-phase glycan isolation for glycomics analysis.

PubMed

Yang, Shuang; Zhang, Hui

2012-12-01

Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for glycomics analysis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activation of a Ca(2+)-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain

NASA Technical Reports Server (NTRS)

Huang, J. F.; Teyton, L.; Harper, J. F.; Evans, M. L. (Principal Investigator)

1996-01-01

Ca(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD). The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor. To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no. L14771) was made as a fusion protein in Escherichia coli. We show here that a truncated CDPK lacking a CaM-LD (e.g. mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM). We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction. When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses. When the junction and CaM-LD are tethered in a single polypeptide (e.g. in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g. the tethered CaM-LD cannot bind a separate junction). A mutation which disrupts the putative CaM-LD binding sequence (e.g. substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding. This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation. Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism. CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide.

A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

PubMed

Carbonetti, Sara; Oliver, Brian G; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Sack, Brandon; Bergl, Emilee; Kappe, Stefan H I; Sather, D Noah

2017-09-01

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

PRIC320, a transcription coactivator, isolated from peroxisome proliferator-binding protein complex.

PubMed

Surapureddi, Sailesh; Viswakarma, Navin; Yu, Songtao; Guo, Dongsheng; Rao, M Sambasiva; Reddy, Janardan K

2006-05-05

Ciprofibrate, a potent peroxisome proliferator, induces pleiotropic responses in liver by activating peroxisome proliferator-activated receptor alpha (PPARalpha), a nuclear receptor. Transcriptional regulation by liganded nuclear receptors involves the participation of coregulators that form multiprotein complexes possibly to achieve cell and gene specific transcription. SDS-PAGE and matrix-assisted laser desorption/ionization reflection time-of-flight mass spectrometric analyses of ciprofibrate-binding proteins from liver nuclear extracts obtained using ciprofibrate-Sepharose affinity matrix resulted in the identification of a new high molecular weight nuclear receptor coactivator, which we designated PRIC320. The full-length human cDNA encoding this protein has an open-reading frame that codes for a 320kDa protein containing 2882 amino acids. PRIC320 contains five LXXLL signature motifs that mediate interaction with nuclear receptors. PRIC320 binds avidly to nuclear receptors PPARalpha, CAR, ERalpha, and RXR, but only minimally with PPARgamma. PRIC320 also interacts with transcription cofactors CBP, PRIP, and PBP. Immunoprecipitation-immunoblotting as well as cellular localization studies confirmed the interaction between PPARalpha and PRIC320. PRIC320 acts as a transcription coactivator by stimulating PPARalpha-mediated transcription. We conclude that ciprofibrate, a PPARalpha ligand, binds a multiprotein complex and PRIC320 cloned from this complex functions as a nuclear receptor coactivator.

Identification of Bacillus thuringiensis Cry1AbMod binding-proteins from Spodoptera frugiperda.

PubMed

Martínez de Castro, Diana L; García-Gómez, Blanca I; Gómez, Isabel; Bravo, Alejandra; Soberón, Mario

2017-12-01

Bacillus thuringiensis Cry toxins are currently used for pest control in transgenic crops but evolution of resistance by the insect pests threatens the use of this technology. The Cry1AbMod toxin was engineered to lack the alpha helix-1 of the parental Cry1Ab toxin and was shown to counter resistance to Cry1Ab and Cry1Ac toxins in different insect species including the fall armyworm Spodoptera frugiperda. In addition, Cry1AbMod showed enhanced toxicity to Cry1Ab-susceptible S. frugiperda populations. To gain insights into the mechanisms of this Cry1AbMod-enhanced toxicity, we isolated the Cry1AbMod toxin binding proteins from S. frugiperda brush border membrane vesicles (BBMV), which were identified by pull-down assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS results indicated that Cry1AbMod toxin could bind to four classes of aminopeptidase (N1, N3, N4 y N5) and actin, with the highest amino acid sequence coverage acquired for APN 1 and APN4. In addition to these proteins, we found other proteins not previously described as Cry toxin binding proteins. This is the first report that suggests the interaction between Cry1AbMod and APN in S. frugiperda. Copyright © 2017 Elsevier Inc. All rights reserved.

Identifying Virulence-Associated Genes Using Transcriptomic and Proteomic Association Analyses of the Plant Parasitic Nematode Bursaphelenchus mucronatus.

PubMed

Zhou, Lifeng; Chen, Fengmao; Pan, Hongyang; Ye, Jianren; Dong, Xuejiao; Li, Chunyan; Lin, Fengling

2016-09-07

Bursaphelenchus mucronatus (B. mucronatus) isolates that originate from different regions may vary in their virulence, but their virulence-associated genes and proteins are poorly understood. Thus, we conducted an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic data of highly and weakly virulent B. mucronatus isolates during the pathogenic processes. Approximately 40,000 annotated unigenes and 5000 proteins were gained from the isolates. When we matched all of the proteins with their detected transcripts, a low correlation coefficient of r = 0.138 was found, indicating probable post-transcriptional gene regulation involved in the pathogenic processes. A functional analysis showed that five differentially expressed proteins which were all highly expressed in the highly virulent isolate were involved in the pathogenic processes of nematodes. Peroxiredoxin, fatty acid- and retinol-binding protein, and glutathione peroxidase relate to resistance against plant defence responses, while β-1,4-endoglucanase and expansin are associated with the breakdown of plant cell walls. Thus, the pathogenesis of B. mucronatus depends on its successful survival in host plants. Our work adds to the understanding of B. mucronatus' pathogenesis, and will aid in controlling B. mucronatus and other pinewood nematode species complexes in the future.

Overcoming the heterologous bias: An in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata

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

Puri, Nidhi; Manoharlal, Raman; Sharma, Monika

2011-01-07

Research highlights: {yields} First report to demonstrate an in vivo expression system of an ABC multidrug transporter CgCdr1p of C. glabrata. {yields} First report on the structure and functional characterization of CgCdr1p. {yields} Functional conservation of divergent but typical residues of CgCdr1p. {yields} CgCdr1p elicits promiscuity towards substrates and has a large drug binding pocket with overlapping specificities. -- Abstract: We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplishedmore » by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the arte-factual concerns encountered in using heterologous systems are totally excluded.« less

Characterization and Enhanced Processing of Soluble, Oligomeric gp140 Envelope Glycoproteins Derived from Human Immunodeficiency Virus Type-1 Primary Isolates

DTIC Science & Technology

2001-05-01

isolates could retain gp120 in an oligomer. A large scale purification scheme was developed using lentil lectin affinity and size exclusion...34 e. Western blot analysis……………………………………………… 35 f. Large scale protein expression and purification…………………... 35 g. Metabolic labeling, size...isolate HIV-1 Env………... 60 c. Large scale antigen preparation and analysis……………………… 67 d. Cleaved, soluble crosslinked primary isolate Env binds

Isolation of Llama Antibody Fragments for Prevention of Dandruff by Phage Display in Shampoo

PubMed Central

Dolk, Edward; van der Vaart, Marcel; Lutje Hulsik, David; Vriend, Gert; de Haard, Hans; Spinelli, Silvia; Cambillau, Christian; Frenken, Leon; Verrips, Theo

2005-01-01

As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain antibody fragments (VHHs) can be extended to very harsh conditions, such as the presence of shampoo containing nonionic and anionic surfactants. We selected several VHHs that bind to the cell wall protein Malf1 of M. furfur, a fungus implicated in causing dandruff. In addition to high stability in the presence of shampoo, these VHHs are also stable under other denaturing conditions, such as high urea concentrations. Many of the stable VHHs were found to contain arginine at position 44. Replacement of the native amino acid at position 44 with arginine in the most stable VHH that lacked this arginine resulted in a dramatic further increase in the stability. The combination of the unique properties of VHHs together with applied phage display and protein engineering is a powerful method for obtaining highly stable VHHs that can be used in a wide range of applications. PMID:15640220

High affinity ligands from in vitro selection: Complex targets

PubMed Central

Morris, Kevin N.; Jensen, Kirk B.; Julin, Carol M.; Weil, Michael; Gold, Larry

1998-01-01

Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems. PMID:9501188

Isolation and biochemical characterization of an antifungal peptide from Amaranthus hypochondriacus seeds.

PubMed

Rivillas-Acevedo, Lina A; Soriano-García, Manuel

2007-12-12

An antifungal peptide, Ay-AMP, was isolated from Amaranthus hypochondriacus seeds by acidic extraction and then purified by reverse-phase high-pressure liquid chromatography. The molecular mass of this peptide, as determined by mass spectrometry, is 3184 Da. The peptide belongs to the superfamily of chitin-binding proteins, containing a single cysteine/glycine-rich chitin-binding domain, and it was found that Ay-AMP degrades chitin. Ay-AMP inhibits the growth, at very low doses, of different pathogenic fungi, such as Candida albicans, Trichoderma sp., Fusarium solani, Penicillium chrysogenum, Geotrichum candidum, Aspergillus candidus, Aspergillus schraceus, and Alternaria alternata. Ay-AMP is very resistant to the effect of proteases and heating; however, it showed an antagonistic effect with CaCl2 and KCl.

Enhanced sampling of glutamate receptor ligand-binding domains.

PubMed

Lau, Albert Y

2018-04-14

The majority of excitatory synaptic transmission in the central nervous system is mediated by ionotropic glutamate receptors (iGluRs). These membrane-bound protein assemblies consist of modular domains that can be genetically isolated and expressed, which has resulted in a plethora of crystal structures of individual domains in different conformations bound to different ligands. These structures have presented opportunities for molecular dynamics (MD) simulation studies. To examine the free energies that govern molecular behavior, simulation strategies and algorithms have been developed, collectively called enhanced sampling methods This review focuses on the use of enhanced sampling MD simulations of isolated iGluR ligand-binding domains to characterize thermodynamic properties important to receptor function. Copyright © 2018 Elsevier B.V. All rights reserved.

Optimisation of plant protein and transglutaminase content in novel beef restructured steaks for older adults by central composite design.

PubMed

Baugreet, Sephora; Kerry, Joseph P; Brodkorb, André; Gomez, Carolina; Auty, Mark; Allen, Paul; Hamill, Ruth M

2018-08-01

With the goal of optimising a protein-enriched restructured beef steak targeted at the nutritional and chemosensory requirements of older adults, technological performance of thirty formulations, containing plant-based ingredients, pea protein isolate (PPI), rice protein (RP) and lentil flour (LF) with transglutaminase (TG) to enhance binding of meat pieces, were analysed. Maximal protein content of 28% in cooked product was achieved with PPI, RP and LF. Binding strength was primarily affected by TG, while textural parameters were improved with LF inclusion. Optimal formulation (F) to obtain a protein-enriched steak with lowest hardness values was achieved with TG (2%), PPI (8%), RP (9.35%) and LF (4%). F, F1S (optimal formulation 1 with added seasoning) and control restructured products (not containing plant proteins or seasonings) were scored by 120 consumers' aged over-65 years. Controls were most preferred (P < .05), while F1S were least liked by the older consumers. Consumer testing suggests further refinement and optimisation of restructured products with plant proteins should be undertaken. Copyright © 2018 Elsevier Ltd. All rights reserved.

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

PubMed Central

Barros, Marilia; Nanda, Hirsh

2016-01-01

ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA. PMID:26912608

Interaction of LY171883 and other peroxisome proliferators with fatty-acid-binding protein isolated from rat liver.

PubMed Central

Cannon, J R; Eacho, P I

1991-01-01

Fatty-acid-binding protein (FABP) is a 14 kDa protein found in hepatic cytosol which binds and transports fatty acids and other hydrophobic ligands throughout the cell. The purpose of this investigation was to determine whether LY171883, a leukotriene D4 antagonist, and other peroxisome proliferators bind to FABP and displace an endogenous fatty acid. [3H]Oleic acid was used to monitor the elution of FABP during chromatographic purification. [14C]LY171883 had a similar elution profile when substituted in the purification, indicating a common interaction with FABP. LY171883 and its structural analogue, LY189585, as well as the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate, bezafibrate and WY14,643, displaced [3H]oleic acid binding to FABP. Analogues of LY171883 that do not induce peroxisome proliferation only weakly displaced oleate binding. [3H]Ly171883 bound directly to FABP with a Kd of 10.8 microM, compared with a Kd of 0.96 microM for [3H]oleate. LY171883 binding was inhibited by LY189585, clofibric acid, ciprofibrate and bezafibrate. These findings demonstrate that peroxisome proliferators, presumably due to their structural similarity to fatty acids, are able to bind to FABP and displace an endogenous ligand from its binding site. Interaction of peroxisome proliferators with FABP may be involved in perturbations of fatty acid metabolism caused by these agents as well as in the development of the pleiotropic response of peroxisome proliferation. Images Fig. 2. PMID:1747111

Platyphylloside Isolated From Betula platyphylla Inhibit Adipocyte Differentiation and Induce Lipolysis Via Regulating Adipokines Including PPARγ in 3T3-L1 Cells

PubMed Central

Lee, Mina; Sung, Sang Hyun

2016-01-01

Background: Obesity causes or aggravates many health problems, both independently and in association with several pathological disorders, including Type II diabetes, hypertension, atherosclerosis, and cancer. Therefore, we screened small compounds isolated from natural products for the development of anti-obesity drugs. Objective: The purpose of this study was to investigate the anti-adipogenic activities of platyphylloside, diarylheptanoid isolated from Betula platyphylla, which was selected based on the screening using 3T3-L1 cells. Materials and Methods: To evaluate the inhibition of adipocyte differentiation and lipolysis, lipid contents of BPP on were measured using Oil Red O staining in 3T3-L1 cells. The mRNA and protein expression levels of various adipokines were measured by Quantitative real-time PCR and Western blotting analysis, respectively. Results: Platyphylloside showed significant inhibitory activity on adipocyte differentiation in 3T3-L1 cells and suppressed adipocyte differentiation even in the presence of troglitazone, a PPARγ agonist. Platyphylloside might suppress adipocyte differentiation through PPARγ, C/EBPα, and SREBP1-induced adipogenesis, which is synergistically associated with downstream adipocyte-specific gene promoters such as aP2, FAS, SCD-1, LPL, and Adiponectin. In addition, platyphylloside affected lipolysis by down-regulating perilipin and HSL and up-regulating TNFα. Conclusion: Taken together, the results reveal that platyphylloside has anti-adipogenic activity and highlight its potential in the prevention and treatment of obesity. SUMMARY The extract of B. platyphylla bark and its isolate, BPP, had anti-adipogenic activity in 3T3-L1 cells via suppression of adipocyte differentiation from preadipocytes.Treatment with BPP significantly down-regulated the expression of PPARγ, C/EBP, C/EBPβ, C/EBPδ, SREBP1c, SCD-1, FAS, aP2 and LPL.BPP induced a lipolytic response in mature adipocytes via up-regulation krof TNFá and down-regulation of HSL, perilipin, PPARγ, PDE3B, and Gia1.BPP is a novel potential agent in the prevention and treatment of obesity through its anti-adipogenic activities and lipolysis. Abbreviations used: DMEM: Dulbecco's modified Eagle's medium, FBS: fetal bovine serum, ORO: Oil Red O, PBS: phosphate buffered saline, RT: room temperature, PPAR: peroxisome proliferator-activated receptor, C/EBP: CCAAT/enhancer-binding protein, SREBP1: sterol regulatory element binding protein 1, SCD-1: steroyl-coenzyme A desaturase 1, LPL: lipoprotein lipase, aP2: adipocyte fatty acid binding protein, FAS: fatty acid synthase, HSL: hormone sensitive lipase, Giα1: GPT binding protein, PDE3B: phosphodiesterase 3B, TNFα: tumor necrosis factor α, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, SD: standard deviation, EGCG: epigallocatechin-3-gallate, TZD: thiazolidinediones PMID:27867269

The ATPase domain of the large terminase protein, gp17, from bacteriophage T4 binds DNA: implications to the DNA packaging mechanism.

PubMed

Alam, Tanfis I; Rao, Venigalla B

2008-03-07

Translocation of double-stranded DNA into a preformed capsid by tailed bacteriophages is driven by powerful motors assembled at the special portal vertex. The motor is thought to drive processive cycles of DNA binding, movement, and release to package the viral genome. In phage T4, there is evidence that the large terminase protein, gene product 17 (gp17), assembles into a multisubunit motor and translocates DNA by an inchworm mechanism. gp17 consists of two domains; an N-terminal ATPase domain (amino acids 1-360) that powers translocation of DNA, and a C-terminal nuclease domain (amino acids 361-610) that cuts concatemeric DNA to generate a headful-size viral genome. While the functional motifs of ATPase and nuclease have been well defined and the ATPase atomic structure has been solved, the DNA binding motif(s) responsible for viral DNA recognition, cutting, and translocation are unknown. Here we report the first evidence for the presence of a double-stranded DNA binding activity in the gp17 ATPase domain. Binding to DNA is sensitive to Mg(2+) and salt, but not the type of DNA used. DNA fragments as short as 20 bp can bind to the ATPase but preferential binding was observed to DNA greater than 1 kb. A high molecular weight ATPase-DNA complex was isolated by gel filtration, suggesting oligomerization of ATPase following DNA interaction. DNA binding was not observed with the full-length gp17, or the C-terminal nuclease domain. The small terminase protein, gp16, inhibited DNA binding, which was further accentuated by ATP. The presence of a DNA binding site in the ATPase domain and its binding properties implicate a role in the DNA packaging mechanism.

Isoform composition and stoichiometry of the approx. 90-kDa heat shock protein associated with glucocorticoid receptors

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

Mendel, D.B.; Orti, E.

1988-05-15

The authors observed that the approx. 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approx. 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approx. 90-kDa heat shock protein. The observation that TSTA and the approx. 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested thatmore » the doublet observed is also due to the existence of two isoforms. They have therefore conducted this study to determine whether TSTA and the approx. 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approx. 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. They used the BuGr1 and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approx. 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with (/sup 35/S)methionine to metabolically label proteins to steady state. The long-term metabolic labeling approach has also enabled them to directly determine that the purified non-activated glucocorticoid receptor contains a single steroid-binding protein and two approx. 90-kDa non-steroid-binding subunits. The consistency with which a approx. 1:2 stoichiometric ratio of steroid binding to approx. 90-kDa protein is observed supports the view that the approx. 90-kDa heat shock protein is a true component of nonactivated glucocorticoid-receptor complexes.« less

NF-kappaB binds to a polymorphic repressor element in the MMP-3 promoter.

PubMed

Borghaei, Ruth C; Rawlings, P Lyle; Javadi, Masoud; Woloshin, Joanna

2004-03-26

A 5T/6T polymorphic site in the matrix metalloproteinase-3 (MMP-3) promoter has been identified as a repressor element involved in inhibiting induction of MMP-3 transcription by interleukin 1; and the 6T allele has been associated with decreased expression of MMP-3 as compared to the 5T allele. Zinc-binding protein-89 (ZBP-89) was cloned from a yeast one-hybrid assay via its ability to interact with this site, but when the protein was over-expressed, it resulted in activation of the MMP-3 promoter rather than repression. Here we show that in nuclear extracts isolated from human gingival fibroblasts stimulated with IL-1, this site is bound by p50 and p65 components of NF-kappaB in addition to ZBP-89, and that recombinant p50 binds preferentially to the 6T binding site. These results are consistent with a role for NF-kappaB in limiting the cytokine induced expression of MMP-3.

Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

DOEpatents

Freimuth, Paul I.

2010-04-06

The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

Characterization of an RNA aptamer against HPV-16 L1 virus-like particles.

PubMed

Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores; Alvarez-Salas, Luis Marat

2014-10-01

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3'end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection.

Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles

PubMed Central

Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores

2014-01-01

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3′end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection. PMID:25111024

Acquisition of Complement Inhibitor Serine Protease Factor I and Its Cofactors C4b-Binding Protein and Factor H by Prevotella intermedia

PubMed Central

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M.

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with 125I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases. PMID:22514678

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

PubMed

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

Influence of E. coli endotoxin on ACTH induced adrenal cell steroidogenesis.

PubMed

Garcia, R; Viloria, M D; Municio, A M

1985-03-01

The effect of endotoxin (lipopolysaccharide from E. coli) on isolated adrenocortical cells was examined. Lipopolysaccharide decreased the ACTH-induced steroidogenesis. This effect was shown by all corticotropin concentrations studied, and the longer the incubation time, the higher the effect produced. The rate of decrease of ACTH-induced steroidogenesis was dependent on the concentration of lipopolysaccharide in the medium. Binding of [125I]ACTH to adrenocortical cells was modified by lipopolysaccharide; this modification was related to a decrease of the ACTH-induced steroidogenesis. This effect supports the hypothesis of a direct interaction between lipopolysaccharide and the cell membrane with a concomitant distortion of the cell surface affecting the ACTH receptor sites of their environment. [14C]Lipopolysaccharide binds to isolated adrenocortical cells. Binding specificity was investigated by competitive experiments in the presence of various types of endotoxins, polypeptide hormones and proteins. Unlabelled lipopolysaccharide from the same bacterial strain and isolated under identical conditions than the labelled lipopolysaccharide exerted the strongest inhibitory activity. Unlabelled lipopolysaccharide of various strains different from that originating the labelled lipopolysaccharide exerted the less displacement. It would imply a certain kind of specificity but the decrease in the binding of lipopolysaccharide produced by ACTH and glucagon suggests the existence of non-specific interactions between lipopolysaccharide and cell membrane.

Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli.

PubMed Central

Houston, L S; Cook, R G; Norris, S J

1990-01-01

A native structure containing the major 60-kilodalton common antigen polypeptide (designated TpN60) was isolated from Treponema pallidum subsp. pallidum (Nichols strain) through a combination of differential centrifugation and sucrose density gradient sedimentation. Gel filtration chromatography indicated that this structure is a high-molecular-weight homo-oligomer of TpN60. Antisera to TpN60 reacted with the groEL polypeptide of Escherichia coli, as determined by immunoperoxidase staining of two-dimensional electroblots. Electron microscopy of the isolated complex revealed a ringlike structure with a diameter of approximately 16 nm which was very similar in appearance to the groEL protein. Comparison of the N-terminal amino acid sequence of TpN60 with the deduced sequences of the E. coli groEL protein, related chaperonin proteins from mycobacteria and Coxiella burnetti, the hsp60 protein of Saccharomyces cerevisiae, the wheat ribulose bisphosphate carboxylase-oxygenase-subunit-binding protein (alpha subunit), and the human P1 mitochondrial protein indicated sequence identity at 8 of 22 to 10 of 22 residues (36 to 45% identity). We conclude that the oligomer of TpN60 is homologous to the groEL protein and related chaperonins found in a wide variety of procaryotes and eucaryotes and thus may represent a heat shock protein involved in protein folding and assembly. Images PMID:1971618

C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

PubMed

Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

2015-09-11

In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The type III effector HsvG of the gall-forming Pantoea agglomerans mediates expression of the host gene HSVGT.

PubMed

Nissan, Gal; Manulis-Sasson, Shulamit; Chalupowicz, Laura; Teper, Doron; Yeheskel, Adva; Pasmanik-Chor, Metsada; Sessa, Guido; Barash, Isaac

2012-02-01

The type III effector HsvG of the gall-forming Pantoea agglomerans pv. gypsophilae is a DNA-binding protein that is imported to the host nucleus and involved in host specificity. The DNA-binding region of HsvG was delineated to 266 amino acids located within a secondary structure region near the N-terminus of the protein but did not display any homology to canonical DNA-binding motifs. A binding site selection procedure was used to isolate a target gene of HsvG, named HSVGT, in Gypsophila paniculata. HSVGT is a predicted acidic protein of the DnaJ family with 244 amino acids. It harbors characteristic conserved motifs of a eukaryotic transcription factor, including a bipartite nuclear localization signal, zinc finger, and leucine zipper DNA-binding motifs. Quantitative real-time polymerase chain reaction analysis demonstrated that HSVGT transcription is specifically induced in planta within 2 h after inoculation with the wild-type P. agglomerans pv. gypsophilae compared with the hsvG mutant. Induction of HSVGT reached a peak of sixfold at 4 h after inoculation and progressively declined thereafter. Gel-shift assay demonstrated that HsvG binds to the HSVGT promoter, indicating that HSVGT is a direct target of HsvG. Our results support the hypothesis that HsvG functions as a transcription factor in gypsophila.

Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

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

Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importancemore » of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.« less

Mutations in Elongation Factor Ef-1α Affect the Frequency of Frameshifting and Amino Acid Misincorporation in Saccharomyces Cerevisiae

PubMed Central

Sandbaken, M. G.; Culbertson, M. R.

1988-01-01

A mutational analysis of the eukaryotic elongation factor EF-1α indicates that this protein functions to limit the frequency of errors during genetic code translation. We found that both amino acid misincorporation and reading frame errors are controlled by EF-1α. In order to examine the function of this protein, the TEF2 gene, which encodes EF-1α in Saccharomyces cerevisiae, was mutagenized in vitro with hydroxylamine. Sixteen independent TEF2 alleles were isolated by their ability to suppress frameshift mutations. DNA sequence analysis identified eight different sites in the EF-1α protein that elevate the frequency of mistranslation when mutated. These sites are located in two different regions of the protein. Amino acid substitutions located in or near the GTP-binding and hydrolysis domain of the protein cause suppression of frameshift and nonsense mutations. These mutations may effect mistranslation by altering the binding or hydrolysis of GTP. Amino acid substitutions located adjacent to a putative aminoacyl-tRNA binding region also suppress frameshift and nonsense mutations. These mutations may alter the binding of aminoacyl-tRNA by EF-1α. The identification of frameshift and nonsense suppressor mutations in EF-1α indicates a role for this protein in limiting amino acid misincorporation and reading frame errors. We suggest that these types of errors are controlled by a common mechanism or closely related mechanisms. PMID:3066688

Karyopherin α 3 and karyopherin α 4 proteins mediate the nuclear import of methyl-CpG binding protein 2.

PubMed

Baker, Steven Andrew; Lombardi, Laura Marie; Zoghbi, Huda Yahya

2015-09-11

Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein with important roles in regulating chromatin structure and gene expression, and mutations in MECP2 cause Rett syndrome (RTT). Within the MeCP2 protein sequence, the nuclear localization signal (NLS) is reported to reside between amino acids 255-271, and certain RTT-causing mutations overlap with the MeCP2 NLS, suggesting that they may alter nuclear localization. One such mutation, R270X, is predicted to interfere with the localization of MeCP2, but recent in vivo studies have demonstrated that this mutant remains entirely nuclear. To clarify the mechanism of MeCP2 nuclear import, we isolated proteins that interact with the NLS and identified karyopherin α 3 (KPNA3 or Kap-α3) and karyopherin α 4 (KPNA4 or Kap-α4) as key binding partners of MeCP2. MeCP2-R270X did not interact with KPNA4, consistent with a requirement for an intact NLS in this interaction. However, this mutant retains binding to KPNA3, accounting for the normal localization of MeCP2-R270X to the nucleus. These data provide a mechanism for MeCP2 nuclear import and have implications for the design of therapeutics aimed at modulating the function of MeCP2 in RTT patients. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - G sub i complex

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

Rollins, T.E.; Siciliano, S.; Kobayashi, S.

1991-02-01

The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K{sub d} of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42,more » 40, and 36 kDa, which were determined to be the C5a-binding subunit and the {alpha} and {beta} subunits of G{sub i}, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.« less

IgG red blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on red blood cell membrane band 3 glycoprotein

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

Victoria, E.J.; Pierce, S.W.; Branks, M.J.

1990-01-01

Red blood cell (RBC) autoantibodies from patients with IgG warm-type autoimmune hemolytic anemia were labeled with iodine 125 and their RBC binding behavior characterized. Epitope-bearing RBC membrane polypeptides were identified after autoantibody immunoprecipitation of labeled membranes and immunoblotting. Immunoaffinity isolation of labeled membrane proteins with 12 different IgG hemolytic autoantibodies with protein A-agarose revealed a major polypeptide at Mr 95 to 110 kd, which coelectrophoresed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with a membrane component isolated with sheep IgG anti-band 3. Immunoprecipitation studies with chymotrypsinized RBCs resulted in the recovery of two labeled membrane polypeptides with molecular weights characteristically resulting frommore » the chymotryptic fragmentation of band 3. Immunoblotting with sheep IgG anti-band 3 of the immunoprecipitated polypeptides confirmed that hemolytic autoantibody binding led to recovery of band 3 or its fragments. Two 125I-labeled IgG hemolytic autoantibodies showed binding behavior consistent with epitope localization on band 3. The labeled RBC autoantibodies bound immunospecifically to all types of human RBC tested, including those of rare Rh type (Rh-null, D--) at a site density of approximately 10(6) per RBC. The 125I-IgG in two labeled autoantibodies was 84% and 92% adsorbable by human and higher nonhuman primate RBCs. Antigen-negative animal RBC bound less than 10%, consistent with immunospecific RBC binding. IgG-1 was the major subclass in five autoantibodies tested; one of six fixed complement; and autoantibody IgG appeared polyclonal by isoelectric focusing. We conclude that IgG eluted from RBCs of patients with autoimmune hemolytic anemia consists predominantly of a single totally RBC-adsorbable antibody population that binds to antigenic determinants on band 3.« less

Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices.

PubMed

Xiong, Yijia; Ford, Nicole R; Hecht, Karen A; Roesijadi, Guritno; Squier, Thomas C

2016-05-18

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.

PubMed

Machida, Kazuya; Liu, Bernard

2017-01-01

Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

PubMed

Yeliseev, Alexei; Zoubak, Lioudmila; Schmidt, Thomas G M

2017-03-01

Human cannabinoid receptor CB 2 belongs to the class A of G protein-coupled receptor (GPCR). CB 2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB 2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB 2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB 2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB 2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB 2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB 2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies. Published by Elsevier Inc.

VOZ; isolation and characterization of novel vascular plant transcription factors with a one-zinc finger from Arabidopsis thaliana.

PubMed

Mitsuda, Nobutaka; Hisabori, Toru; Takeyasu, Kunio; Sato, Masa H

2004-07-01

A 38-bp pollen-specific cis-acting region of the AVP1 gene is involved in the expression of the Arabidopsis thaliana V-PPase during pollen development. Here, we report the isolation and structural characterization of AtVOZ1 and AtVOZ2, novel transcription factors that bind to the 38-bp cis-acting region of A. thaliana V-PPase gene, AVP1. AtVOZ1 and AtVOZ2 show 53% amino acid sequence similarity. Homologs of AtVOZ1 and AtVOZ2 are found in various vascular plants as well as a moss, Physcomitrella patens. Promoter-beta-glucuronidase reporter analysis shows that AtVOZ1 is specifically expressed in the phloem tissue and AtVOZ2 is strongly expressed in the root. In vivo transient effector-reporter analysis in A. thaliana suspension-cultured cells demonstrates that AtVOZ1 and AtVOZ2 function as transcriptional activators in the Arabidopsis cell. Two conserved regions termed Domain-A and Domain-B were identified from an alignment of AtVOZ proteins and their homologs of O. sativa and P. patens. AtVOZ2 binds as a dimer to the specific palindromic sequence, GCGTNx7ACGC, with Domain-B, which is comprised of a functional novel zinc coordinating motif and a conserved basic region. Domain-B is shown to function as both the DNA-binding and the dimerization domains of AtVOZ2. From highly the conservative nature among all identified VOZ proteins, we conclude that Domain-B is responsible for the DNA binding and dimerization of all VOZ-family proteins and designate it as the VOZ-domain.

Molecular cloning and chromosomal localization of a pseudogene related to the human Acyl-CoA binding protein/diazepam binding inhibitor

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

Gersuk, V.H.; Rose, T.M.; Todaro, G.J.

The acyl-CoA binding protein (ACBP) and the diazepam binding inhibitor (DBI) or endozepine are independent isolates of a single 86-amino-acid, 10-kDa protein. ACBP/DBI is highly conserved between species and has been identified in several diverse organisms, including human, cow, rat, frog, duck, insects, plants, and yeast. Although the genomic locus has not yet been cloned in humans, complementary DNA clones with different 5{prime} ends have been isolated and characterized. These cDNA clones appear to be encoded by a single gene. However, Southern blot analyses, in situ hybridizations, and somatic cell hybrid chromosomal mapping all suggest that there are multiple ACBP/DBI-relatedmore » sequences in the genome. To identify potential members of this gene family, degenerate oligonucleotides corresponding to highly conserved regions of ACBP/DBI were used to screen a human genomic DNA library using the polymerase chain reaction. A novel gene, DBIP1, that is closely related to ACBP/DBI but is clearly distinct was identified. DBIP1 bears extensive sequence homology to ACBP/DBI but lacks the introns predicted by rat and duck genomic sequence studies. A 1-base deletion in the coding region results in a frameshift and, along with the absence of introns and the lack of a detectable transcript, suggests that DBIP1 is a pseudogene. ACBP/DBI has previously been mapped to chromosome 2, although this was recently disputed, and a chromosome 6 location was suggested. We show that ACBP/DBI is correctly placed on chromosome 2 and that the gene identified on chromosome 6 is DBIP1. 33 refs., 3 figs., 1 tab.« less

Novel method for detection of glycogen in cells.

PubMed

Skurat, Alexander V; Segvich, Dyann M; DePaoli-Roach, Anna A; Roach, Peter J

2017-05-01

Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing g lutathione S-transferase, a cM c eptitope and the tbd1 BM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Immobilized OBOC combinatorial bead array to facilitate multiplicative screening.

PubMed

Xiao, Wenwu; Bononi, Fernanda C; Townsend, Jared; Li, Yuanpei; Liu, Ruiwu; Lam, Kit S

2013-07-01

One-bead-one-compound (OBOC) combinatorial library screening has been broadly utilized for the last two decades to identify small molecules, peptides or peptidomimetics targeting variable screening probes such as cell surface receptors, bacteria, protein kinases, phosphatases, proteases etc. In previous screening methods, library beads were suspended in solution and screened against one single probe. Only the positive beads were tracked and isolated for additional screens and finally selected for chemical decoding. During this process, the remaining negative beads were not tracked and discarded. Here we report a novel bead immobilization method such that a bead library array can be conveniently prepared and screened in its entirety, sequentially many times with a series of distinct probes. This method not only allows us to increase the screening efficiency but also permits us to determine the binding profile of each and every library bead against a large number of target receptors. As proof of concept, we serially screened a random OBOC disulfide containing cyclic heptapeptide library with three water soluble dyes as model probes: malachite green, bromocresol purple and indigo carmine. This multiplicative screening approach resulted in a rapid determination of the binding profile of each and every bead respective to each of the three dyes. Beads that interacted with malachite green only, bromocresol purple only, or both indigo carmine and bromocresol purple were isolated, and their peptide sequences were determined with microsequencer. Ultimately, the novel OBOC multiplicative screening approach could play a key role in the enhancement of existing on-bead assays such as whole cell binding, bacteria binding, protein binding, posttranslational modifications etc. with increased efficiency, capacity, and specificity.

Isolation and functional characterization of TIF-IB, a factor that confers promoter specificity to mouse RNA polymerase I.

PubMed

Schnapp, A; Clos, J; Hädelt, W; Schreck, R; Cvekl, A; Grummt, I

1990-03-25

The murine ribosomal gene promoter contains two cis-acting control elements which operate in concert to promote efficient and accurate transcription initiation by RNA polymerase I. The start site proximal core element which is indispensable for promoter recognition by RNA polymerase I (pol I) encompasses sequences from position -39 to -1. An upstream control element (UCE) which is located between nucleotides -142 and -112 stimulates the efficiency of transcription initiation both in vivo and in vitro. Here we report the isolation and functional characterization of a specific rDNA binding protein, the transcription initiation factor TIF-IB, which specifically interacts with the core region of the mouse ribosomal RNA gene promoter. Highly purified TIF-IB complements transcriptional activity in the presence of two other essential initiation factors TIF-IA and TIF-IC. We demonstrate that the binding efficiency of purified TIF-IB to the core promoter is strongly enhanced by the presence in cis of the UCE. This positive effect of upstream sequences on TIF-IB binding is observed throughout the purification procedure suggesting that the synergistic action of the two distant promoter elements is not mediated by a protein different from TIF-IB. Increasing the distance between both control elements still facilitates stable factor binding but eliminates transcriptional activation. The results demonstrate that TIF-IB binding to the rDNA promoter is an essential early step in the assembly of a functional transcription initiation complex. The subsequent interaction of TIF-IB with other auxiliary transcription initiation factors, however, requires the correct spacing between the UCE and the core promoter element.

Intrinsically Disordered Energy Landscapes

PubMed Central

Chebaro, Yassmine; Ballard, Andrew J.; Chakraborty, Debayan; Wales, David J.

2015-01-01

Analysis of an intrinsically disordered protein (IDP) reveals an underlying multifunnel structure for the energy landscape. We suggest that such ‘intrinsically disordered’ landscapes, with a number of very different competing low-energy structures, are likely to characterise IDPs, and provide a useful way to address their properties. In particular, IDPs are present in many cellular protein interaction networks, and several questions arise regarding how they bind to partners. Are conformations resembling the bound structure selected for binding, or does further folding occur on binding the partner in a induced-fit fashion? We focus on the p53 upregulated modulator of apoptosis (PUMA) protein, which adopts an -helical conformation when bound to its partner, and is involved in the activation of apoptosis. Recent experimental evidence shows that folding is not necessary for binding, and supports an induced-fit mechanism. Using a variety of computational approaches we deduce the molecular mechanism behind the instability of the PUMA peptide as a helix in isolation. We find significant barriers between partially folded states and the helix. Our results show that the favoured conformations are molten-globule like, stabilised by charged and hydrophobic contacts, with structures resembling the bound state relatively unpopulated in equilibrium. PMID:25999294

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen

USDA-ARS?s Scientific Manuscript database

Disease resistance (R) genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLRs define the fastest evolving...

Relative quantitative comparisons of the extracellular protein profiles of Staphylococcus aureus UAMS-1 and its sarA, agr, and sarA agr regulatory mutants using one-dimensional polyacrylamide gel electrophoresis and nanocapillary liquid chromatography coupled with tandem mass spectrometry.

PubMed

Jones, Richard C; Deck, Joanna; Edmondson, Ricky D; Hart, Mark E

2008-08-01

One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography coupled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more abundant early on in growth, while proteins known to be secreted into the surrounding milieu were more abundant late in growth. In addition, proteins from spent media and cell lysates of strain UAMS-1 and its isogenic sarA, agr, and sarA agr regulatory mutant strains during exponential growth were identified, and their relative abundances were compared. Extracellular proteins known to be regulated by the global regulators sarA and agr displayed protein levels in accordance with what is known regarding the effects of these regulators. For example, cysteine protease (SspB), endopeptidase (SspA), staphopain (ScpA), and aureolysin (Aur) were higher in abundance in the sarA and sarA agr mutants than in strain UAMS-1. The immunoglobulin G (IgG)-binding protein (Sbi), immunodominant staphylococcal antigen A (IsaA), IgG-binding protein A (Spa), and the heme-iron-binding protein (IsdA) were most abundant in the agr mutant background. Proteins whose abundance was decreased in the sarA mutant included fibrinogen-binding protein (Fib [Efb]), IsaA, lipase 1 and 2, and two proteins identified as putative leukocidin F and S subunits of the two-component leukotoxin family. Collectively, this approach identified 1,263 proteins (matches of two peptides or more) and provided a convenient and reliable way of identifying proteins and comparing their relative abundances.

A dsRNA-binding protein MdDRB1 associated with miRNA biogenesis modifies adventitious rooting and tree architecture in apple.

PubMed

You, Chun-Xiang; Zhao, Qiang; Wang, Xiao-Fei; Xie, Xing-Bin; Feng, Xiao-Ming; Zhao, Ling-Ling; Shu, Huai-Rui; Hao, Yu-Jin

2014-02-01

Although numerous miRNAs have been already isolated from fruit trees, knowledge about miRNA biogenesis is largely unknown in fruit trees. Double-strand RNA-binding (DRB) protein plays an important role in miRNA processing and maturation; however, its role in the regulation of economically important traits is not clear yet in fruit trees. EST blast and RACE amplification were performed to isolate apple MdDRB1 gene. Following expression analysis, RNA binding and protein interaction assays, MdDRB1 was transformed into apple callus and in vitro tissue cultures to characterize the functions of MdDRB1 in miRNA biogenesis, adventitious rooting, leaf development and tree growth habit. MdDRB1 contained two highly conserved DRB domains. Its transcripts existed in all tissues tested and are induced by hormones. It bound to double-strand RNAs and interacted with AtDCL1 (Dicer-Like 1) and MdDCL1. Chip assay indicated its role in miRNA biogenesis. Transgenic analysis showed that MdDRB1 controls adventitious rooting, leaf curvature and tree architecture by modulating the accumulation of miRNAs and the transcript levels of miRNA target genes. Our results demonstrated that MdDRB1 functions in the miRNA biogenesis in a conserved way and that it is a master regulator in the formation of economically important traits in fruit trees. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Cariogenic properties of Streptococcus mutans clinical isolates with sortase defects.

PubMed

Lapirattanakul, Jinthana; Takashima, Yukiko; Tantivitayakul, Pornpen; Maudcheingka, Thaniya; Leelataweewud, Pattarawadee; Nakano, Kazuhiko; Matsumoto-Nakano, Michiyo

2017-09-01

In Streptococcus mutans, a Gram-positive pathogen of dental caries, several surface proteins are anchored by the activity of sortase enzyme. Although various reports have shown that constructed S. mutans mutants deficient of sortase as well as laboratory reference strains with a sortase gene mutation have low cariogenic potential, no known studies have investigated clinical isolates with sortase defects. Here, we examined the cariogenic properties of S. mutans clinical isolates with sortase defects as well as caries status in humans harboring such defective isolates. Sortase-defective clinical isolates were evaluated for biofilm formation, sucrose-dependent adhesion, stress-induced dextran-dependent aggregation, acid production, and acid tolerance. Additionally, caries indices of subjects possessing such defective isolates were determined. Our in vitro results indicated that biofilm with a lower quantity was formed by sortase-defective as compared to non-defective isolates. Moreover, impairments of sucrose-dependent adhesion and stress-induced dextran-dependent aggregation were found among the isolates with defects, whereas no alterations were seen in regard to acid production or tolerance. Furthermore, glucan-binding protein C, a surface protein anchored by sortase activity, was predominantly detected in culture supernatants of all sortase-defective S. mutans isolates. Although the sortase-defective isolates showed lower cariogenic potential because of a reduction in some cariogenic properties, deft/DMFT indices revealed that all subjects harboring those isolates had caries experience. Our findings suggest the impairment of cariogenic properties in S. mutans clinical isolates with sortase defects, though the detection of these defective isolates seemed not to imply low caries risk in the subjects harboring them. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pomegranate Cultivars: Identification of the New IgE-Binding Protein Pommaclein and Analysis of Antioxidant Variability.

PubMed

Tuppo, Lisa; Alessandri, Claudia; Pasquariello, Maria Silvia; Petriccione, Milena; Giangrieco, Ivana; Tamburrini, Maurizio; Mari, Adriano; Ciardiello, Maria Antonietta

2017-04-05

The consumption of pomegranate is increasing as it is considered a health-promoting food. Nevertheless, it can trigger allergic reactions, sometimes severe. The LTP Pun g 1 is the only pomegranate allergen so far reported. Based on preliminary clinical observations, the main aim of this study was the investigation of still unknown allergens contained in this fruit. Pommaclein, a homologue of peamaclein, the peach allergen Pru p 7, was isolated, identified by protein sequencing, and characterized as an IgE-binding protein by different test systems. RP-HPLC protein profiles revealed significant variations of LTP and pommaclein content in the red pulp of selected cultivars and accessions. Conversely, the mesocarp appeared free of proteins and much richer in antioxidants. In conclusion, a new allergen has been identified, and it could contribute to improving allergy diagnosis. The study highlights that pomegranate mesocarp could represent a rich and safe source of nutraceuticals also for allergic subjects.

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

PubMed Central

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

1979-01-01

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

Solubilization of a membrane protein by combinatorial supercharging.

PubMed

Hajduczki, Agnes; Majumdar, Sudipta; Fricke, Marie; Brown, Isola A M; Weiss, Gregory A

2011-04-15

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

Coating cells with colloidal silica for high yield isolation of plasma membrane sheets and identification of transmembrane proteins.

PubMed

Chaney, L K; Jacobson, B S

1983-08-25

Plasma membrane (PM) can be isolated by binding to a positively charged solid support. Using this concept, we have developed a novel method of PM isolation using cationic colloidal silica. The method is designed for the comparative study of various physiological states of PM and for transbilayer protein mapping. The procedure consists of coating intact cells with a dense pellicle of silica particles and polyanion. Since cells remain intact during pellicle formation, the external face of the PM is selectively coated. The pellicle greatly enhances PM density and stabilizes it against vesiculation or lateral reorientation. Upon cell lysis, large open sheets of PM are rapidly isolated by centrifugation. PM from Dictyostelium discoideum was prepared by this method. Marker enzymes, cell surface labeling and microscopy demonstrate that the PM was isolated in high yield (70-80%) with a 10-17-fold purification and only low levels of cytoplasmic contamination. The pellicle remains intact during cell lysis and membrane isolation, shielding the external surface of the membranes up to 92% from chemical or enzymatic attack. The PM can thus be labeled selectively from inside and/or outside. Transmembrane proteins were identified in Dictyostelium PM by means of lactoperoxidase iodination and autoradiography.

Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters

DOE PAGES

Seppala, Susanna; Solomon, Kevin V.; Gilmore, Sean P.; ...

2016-12-20

Here, engineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive andmore » rather extreme environment. Using sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains. In conclusion, we report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes produce.« less

Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters.

PubMed

Seppälä, Susanna; Solomon, Kevin V; Gilmore, Sean P; Henske, John K; O'Malley, Michelle A

2016-12-20

Engineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive and rather extreme environment. Using sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains. We report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes produce.

Rat leucine-rich protein binds and activates the promoter of the beta isoform of Ca2+/calmodulin-dependent protein kinase II gene.

PubMed

Ochiai, Nagahiro; Masumoto, Shuji; Sakagami, Hiroyuki; Yoshimura, Yoshiyuki; Yamauchi, Takashi

2007-05-01

We previously found the neuronal cell-type specific promoter and binding partner of the beta isoform of Ca(2+)/calmodulin-dependent protein kinase II (beta CaM kinase II) in rat brain [Donai, H., Morinaga, H., Yamauchi, T., 2001. Genomic organization and neuronal cell type specific promoter activity of beta isoform of Ca(2+)/calmodulin-dependent protein kinase II of rat brain. Mol. Brain Res. 94, 35-47]. In the present study, we purified a protein that binds specifically a promoter region of beta CaM kinase II gene from a nuclear extract of the rat cerebellum using DEAE-cellulose column chromatography, ammonium sulfate fractionation, gel filtration and polyacrylamide gel electrophoresis. The purified protein was identified as rat leucine-rich protein 157 (rLRP157) using tandem mass spectrometry. Then, we prepared its cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) from poly(A)(+)RNA of rat cerebellum. The rLRP157 cDNA was introduced into mouse neuroblastomaxrat glioma hybrid NG108-15 cells, and cells stably expressing rLRP157 (NG/LRP cells) were isolated. Binding of rLRP157 with the promoter sequence was confirmed by electrophoretic mobility shift assay using nuclear extract of NG/LRP cells. A luciferase reporter gene containing a promoter of beta CaM kinase II was transiently expressed in NG/LRP cells. Under the conditions, the promoter activity was enhanced about 2.6-fold in NG/LRP cells as compared with wild-type cells. The expression of rLRP157 mRNA was paralleled with that of beta CaM kinase II in the adult and embryo rat brain detected by in situ hybridization. Nuclear localization of rLRP157 was confirmed using GFP-rLRP157 fusion protein investigated under a confocal microscope. These results indicate that rLRP157 is one of the proteins binding to, and regulating the activity of, the promoter of beta CaM kinase II.

Advantages of Molecular Weight Identification during Native MS Screening.

PubMed

Khan, Ahad; Bresnick, Anne; Cahill, Sean; Girvin, Mark; Almo, Steve; Quinn, Ronald

2018-05-09

Native mass spectrometry detection of ligand-protein complexes allowed rapid detection of natural product binders of apo and calcium-bound S100A4 (a member of the metal binding protein S100 family), T cell/transmembrane, immunoglobulin (Ig), and mucin protein 3, and T cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains precursor protein from extracts and fractions. Based on molecular weight common hits were detected binding to all four proteins. Seven common hits were identified as apigenin 6- C - β - D -glucoside 8- C - α - L -arabinoside, sweroside, 4',5-dihydroxy-7-methoxyflavanone-6- C -rutinoside, loganin acid, 6- C -glucosylnaringenin, biochanin A 7- O -rutinoside and quercetin 3- O -rutinoside. Mass guided isolation and NMR identification of hits confirmed the mass accuracy of the ligand in the ligand-protein MS complexes. Thus, molecular weight ID from ligand-protein complexes by electrospray ionization Fourier transform mass spectrometry allowed rapid dereplication. Native mass spectrometry using electrospray ionization Fourier transform mass spectrometry is a tool for dereplication and metabolomics analysis. Georg Thieme Verlag KG Stuttgart · New York.

YC-1 BINDING TO THE BETA SUBUNIT OF SOLUBLE GUANYLYL CYCLASE OVERCOMES ALLOSTERIC INHIBITION BY THE ALPHA SUBUNIT

PubMed Central

Purohit, Rahul; Fritz, Bradley G.; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L.; Campbell, Eric; Hausrath, Andrew C.; Rassouli-Taylor, Leida; Garcin, Elsa D.; Gage, Matthew J.; Montfort, William R.

2014-01-01

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity. PMID:24328155

Rat Sertoli cells acquire a beta-adrenergic response during primary culture.

PubMed Central

Kierszenbaum, A L; Spruill, W A; White, M G; Tres, L L; Perkins, J P

1985-01-01

Two-dimensional polyacrylamide gel electrophoresis and the radioligand (-)-[125I]iodopindolol (125I-Pin) have been used to study isoproterenol-dependent protein phosphorylation and beta-adrenergic receptor availability, respectively, in cultured Sertoli cells and freshly isolated seminiferous tubular segments of sexually immature and mature rats. Sertoli cells prepared from sexually immature rats show progressive 125I-Pin binding in primary cultures that correlates with isoproterenol-induced cell shape changes, redistribution of immunoreactive vimentin, and phosphorylation of this intermediate filament protein. The development of 125I-Pin binding to Sertoli cell lysates is blocked by cycloheximide. Seminiferous tubules do not show significant isoproterenol-dependent vimentin phosphorylation nor 125I-Pin binding. However, vimentin phosphorylation can be induced by follicle-stimulating hormone or a cyclic nucleotide analog. This study stresses the need for correlating pharmacological-induced responses observed in Sertoli cell primary cultures with those in the intact seminiferous tubule. Images PMID:2984678

Nucleotide-dependent assembly of the peroxisomal receptor export complex

PubMed Central

Grimm, Immanuel; Saffian, Delia; Girzalsky, Wolfgang; Erdmann, Ralf

2016-01-01

Pex1p and Pex6p are two AAA-ATPases required for biogenesis of peroxisomes. Both proteins form a hetero-hexameric complex in an ATP-dependent manner, which has a dual localization in the cytosol and at the peroxisomal membrane. At the peroxisomal membrane, the complex is responsible for the release of the import receptor Pex5p at the end of the matrix protein import cycle. In this study, we analyzed the recruitment of the AAA-complex to its anchor protein Pex15p at the peroxisomal membrane. We show that the AAA-complex is properly assembled even under ADP-conditions and is able to bind efficiently to Pex15p in vivo. We reconstituted binding of the Pex1/6p-complex to Pex15p in vitro and show that Pex6p mediates binding to the cytosolic part of Pex15p via a direct interaction. Analysis of the isolated complex revealed a stoichiometry of Pex1p/Pex6p/Pex15p of 3:3:3, indicating that each Pex6p molecule of the AAA-complex binds Pex15p. Binding of the AAA-complex to Pex15p in particular and to the import machinery in general is stabilized when ATP is bound to the second AAA-domain of Pex6p and its hydrolysis is prevented. The data indicate that receptor release in peroxisomal protein import is associated with a nucleotide-depending Pex1/6p-cycle of Pex15p-binding and release. PMID:26842748

Purification and characterization of an antifungal protein, C-FKBP, from Chinese cabbage.

PubMed

Park, Seong-Cheol; Lee, Jung Ro; Shin, Sun-Oh; Jung, Ji Hyun; Lee, Young Mee; Son, Hyosuk; Park, Yoonkyung; Lee, Sang Yeol; Hahm, Kyung-Soo

2007-06-27

An antifungal protein was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by buffer-soluble extraction and two chromatographic procedures. The results of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the isolated Chinese cabbage protein was identical to human FK506-binding protein (FKBP). A cDNA encoding FKBP was isolated from a Chinese cabbage leaf cDNA library and named C-FKBP. The open reading frame of the gene encoded a 154-amino acid polypeptide. The amino acid sequence of C-FKBP exhibits striking degrees of identity with the corresponding mouse (61%), human (60%), and yeast (56%) proteins. Genomic Southern blot analyses using the full-length C-FKBP cDNA probe revealed a multigene family in the Chinese cabbage genome. The C-FKBP mRNA was highly expressed in vegetative tissues. We also analyzed the antifungal and peptidyl-prolyl cis-trans isomerase activity of recombinant C-FKBP protein expressed in Escherichia coli. This protein inhibited pathogenic fungal strains, including Candida albicans, Botrytis cinerea, Rhizoctonia solani, and Trichoderma viride, whereas it exhibited no activity against E. coli and Staphylococcus aureus. These results suggest that recombinant C-FKBP is an excellent candidate as a lead compound for the development of antifungal agents.

Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities.

PubMed

Parisi, Mónica G; Moreno, Silvia; Fernández, Graciela

2008-04-01

A dual function protein was isolated from Allium sativum bulbs and was characterized. The protein had a molecular mass of 25-26 kDa under non-reducing conditions, whereas two polypeptide chains of 12.5+/-0.5 kDa were observed under reducing conditions. E-64 and leupeptin inhibited the proteolytic activity of the protein, which exhibited characteristics similar to cysteine peptidase. The enzyme exhibited substrate specificity and hydrolyzed natural substrates such as alpha-casein (K(m): 23.0 microM), azocasein, haemoglobin and gelatin. It also showed a high affinity for synthetic peptides such as Cbz-Ala-Arg-Arg-OMe-beta-Nam (K(m): 55.24 microM, k(cat): 0.92 s(-1)). The cysteine peptidase activity showed a remarkable stability after incubation at moderate temperatures (40-50 degrees C) over a pH range of 5.5-6.5. The N-terminus of the protein displayed a 100% sequence similarity to the sequences of a mannose-binding lectin isolated from garlic bulbs. Moreover, the purified protein was retained in the chromatographic column when Con-A Sepharose affinity chromatography was performed and the protein was able to agglutinate trypsin-treated rabbit red cells. Therefore, our results indicate the presence of an additional cysteine peptidase activity on a lectin previously described.

Structural basis for new pattern of conserved amino acid residues related to chitin-binding in the antifungal peptide from the coconut rhinoceros beetle Oryctes rhinoceros.

PubMed

Hemmi, Hikaru; Ishibashi, Jun; Tomie, Tetsuya; Yamakawa, Minoru

2003-06-20

Scarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros is a 36-residue polypeptide that has antifungal activity. The solution structure of scarabaecin has been determined from twodimensional 1H NMR spectroscopic data and hybrid distance geometry-simulated annealing protocol calculation. Based on 492 interproton and 10 hydrogen-bonding distance restraints and 36 dihedral angle restraints, we obtained 20 structures. The average backbone root-mean-square deviation for residues 4-35 is 0.728 +/- 0.217 A from the mean structure. The solution structure consists of a two-stranded antiparallel beta-sheet connected by a type-I beta-turn after a short helical turn. All secondary structures and a conserved disulfide bond are located in the C-terminal half of the peptide, residues 18-36. Overall folding is stabilized by a combination of a disulfide bond, seven hydrogen bonds, and numerous hydrophobic interactions. The structural motif of the C-terminal half shares a significant tertiary structural similarity with chitin-binding domains of plant and invertebrate chitin-binding proteins, even though scarabaecin has no overall sequence similarity to other peptide/polypeptides including chitin-binding proteins. The length of its primary structure, the number of disulfide bonds, and the pattern of conserved functional residues binding to chitin in scarabaecin differ from those of chitin-binding proteins in other invertebrates and plants, suggesting that scarabaecin does not share a common ancestor with them. These results are thought to provide further strong experimental evidence to the hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process.

On the Role of Water Models in Quantifying the Binding Free Energy of Highly Conserved Water Molecules in Proteins: The Case of Concanavalin A.

PubMed

Fadda, Elisa; Woods, Robert J

2011-10-11

The ability of ligands to displace conserved water molecules in protein binding sites is of significant interest in drug design and is particularly pertinent in the case of glycomimetic drugs. This concept was explored in previous work [ Clarke et al. J. Am. Chem. Soc. 2001 , 123 , 12238 - 12247 and Kadirvelraj et al. J. Am. Chem. Soc. 2008 , 130 , 16933 - 16942 ] for a highly conserved water molecule located in the binding site of the prototypic carbohydrate-binding protein Concanavalin A (Con A). A synthetic ligand was designed with the aim of displacing such water. While the synthetic ligand bound to Con A in an analogous manner to that of the natural ligand, crystallographic analysis demonstrated that it did not displace the conserved water. In order to quantify the affinity of this particular water for the Con A surface, we report here the calculated standard binding free energy for this water in both ligand-bound and free Con A, employing three popular water models: TIP3P, TIP4P, and TIP5P. Although each model was developed to perform well in simulations of bulk-phase water, the computed binding energies for the isolated water molecule displayed a high sensitivity to the model. Both molecular dynamics simulation and free energy results indicate that the choice of water model may greatly influence the characterization of surface water molecules as conserved (TIP5P) or not (TIP3P) in protein binding sites, an observation of considerable significance to rational drug design. Structural and theoretical aspects at the basis of the different behaviors are identified and discussed.

The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection.

PubMed

Mingzhang, Rao; Zijun, Zhao; Lixia, Yuan; Jian, Chen; Min, Feng; Jie, Zhang; Ming, Liao; Weisheng, Cao

2018-01-01

A novel avian leukosis viruses (ALV) subgroup named ALV-K was recently isolated from Chinese indigenous chickens which is different from the subgroups (A to E and J) that have previously been reported to infect chickens. More and more ALV-K strains have recently been isolated from local breeds of Chinese chickens. However, there are no more effective diagnostic methods for ALV-K other than virus isolation followed by envelope gene sequencing and comparison. Viral infection can be blocked through expression of the viral receptor-binding protein. In this study, we have engineered a cell line, DF-1/K, that expresses ALV-K env protein and thereby confers resistance to ALV-K infection. DF-1/K can be used in combination with the ALV-K susceptible cell line DF-1 as a specific diagnostic tool for ALV-K and provides a good tool for further research into the molecular mechanisms of interaction between ALV-K env protein and the host cell receptor.

Structural and Binding Properties of Two Paralogous Fatty Acid Binding Proteins of Taenia solium Metacestode

PubMed Central

Yang, Hyun-Jong; Shin, Joo-Ho; Diaz-Camacho, Sylvia Paz; Nawa, Yukifumi; Kang, Insug; Kong, Yoon

2012-01-01

Background Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. Methodology/Principal Findings We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15–95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids and retinol. Conclusions/Significance The divergent biochemical properties, physiological roles and cellular distributions of the TsMFABPs might be one of the critical mechanisms compensating for inadequate de novo FA synthesis. These proteins might exert harmonized or independent roles on lipid assimilation and intracellular signaling. The specialized distribution of retinol in the canal region further implies that cells in this region might differentiate into diverse cell types during metamorphosis into an adult worm. Identification of bioactive systems pertinent to parasitic homeostasis may provide a valuable target for function-related drug design. PMID:23150743

Anti-sulfotyrosine antibodies

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

2009-09-15

The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

Polynucleotides encoding anti-sulfotyrosine antibodies

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

2011-01-11

The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

Rapid isolation of novel FK506 binding proteins from multiple organisms using gDNA and cDNA T7 phage display.

PubMed

Piggott, Andrew M; Kriegel, Alison M; Willows, Robert D; Karuso, Peter

2009-10-01

Reverse chemical proteomics using T7 phage display is a powerful technique for identifying cellular receptors of biologically active small molecules. However, to date this method has generally been limited to cDNA libraries constructed from mRNA isolated from eukaryotes. In this paper, we describe the construction of the first prokaryotic T7 phage display libraries from randomly digested Pseudomonas stutzeri and Vibrio fischeri gDNA, as well as a plant cDNA library from Arabidopsis thaliana. We also describe the use of T7 phage display to identify novel proteins from environmental DNA samples using biotinylated FK506 as a model affinity probe.

Biological features of hepatitis B virus isolates from patients based on full-length genomic analysis.

PubMed

Wen, Yu-Mei; Wang, Yong-Xiang

2009-01-01

The mechanisms for HBV persistence and the pathogenesis of chronic HB have been shown mainly due to defects in host immune responses. However, HBV isolates with different biological features may also contribute to different clinical outcomes and epidemiological implications in viral hepatitis B (HB). This review presents interesting biological features of HBV isolates based on the structural and functional analysis of full-length HBV isolates from various patients. Among isolates from children after failure of HB vaccination, 129L mutant at the 'a' determinant was found with normal binding efficiency to anti-HBs, but with reduced immunogenicity, which could initiate persistent HBV infections. Isolates from fulminant hepatitis (FH) B patients were not all highly replicative, but differences in capacities of anti-HBs induction could be involved in the pathogenesis of FH. The high replicative competency of isolates from hepatocellular carcinoma (HCC) patients could result in enhanced immune-mediated cytopathic effects against HBV viral proteins, and increased transactivating activity by the X protein. The mechanism of a double-spliced variant in enhancing replication of the wild-type virus is presented. The importance of integrating structural and functional analysis to reveal biological features of HBV isolates in viral pathogenesis is discussed.

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

PubMed Central

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

2006-01-01

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

Human lactoferrin binding to Porphyromonas gingivalis, Prevotella intermedia and Prevotella melaninogenica.

PubMed

Kalfas, S; Andersson, M; Edwardsson, S; Forsgren, A; Naidu, A S

1991-12-01

Human isolates of Porphyromonas gingivalis (n = 16), Prevotella intermedia n = 82) and Prevotella melaninogenica (n = 18) from diseased periodontal pockets were examined for interaction with human lactoferrin (HLf) in a standardized 125I-labeled protein binding assay. The highest HLf binding was found in P. intermedia strains, followed by P. gingivalis and P. melaninogenica. Further characterization of the interaction was performed with 1 representative strain from each species. HLf binding to P. gingivalis reached a saturation instantly and was optimal at pH 5.0-6.5. The corresponding values for P. melaninogenica were 90 min and pH 3.0-5.5. The HLf binding to the 2 strains seem to be nonspecific. In contrast, P. intermedia demonstrated specific binding, and a time-saturability within 60 min with an optimal uptake at pH 6.0-7.5. Scatchard analysis implied 45,000 receptors per cell with an affinity constant of 5.5 x 10(-7) M on P. intermedia strain 4H. The binding capacity in all 3 strains was affected by the culture medium. HLf binding components in these strains were susceptible to heat or proteases. Binding was eliminated in P. gingivalis and was enhanced in P. intermedia and P. melaninogenica by periodate treatment. Unlabeled HLf or bovine lactoferrin effectively displaced labeled HLf binding. Various proteins and carbohydrates did not inhibit HLf binding. Our data suggest that HLf binds to these periodontitis-associated species and that this mechanism is distinct from the previously known ligand interactions in oral bacteria.

Immunocytochemical Studies of Neurofibrillary Tangles

PubMed Central

Yen, Shu-Hui C.; Gaskin, Felicia; Terry, Robert D.

1981-01-01

The molecular nature of neurofibrillary tangles of senile dementia of the Alzheimer type (SDAT) was studied by immunoperoxidase and immunofluorescence techniques. Five antiserums, including anti-humanbrain-2-cycle-purified-microtubule-fractions (2 × MT), anti-calf-brain-2 × MT, anti-sea-urchin-egg-tubulin, antibeef-brain-tubulin, and anti-human-brain-neurofilament(NF)-210-kilodalton(kd)-protein were tested for their binding to neurofibrillary tangles. The antihuman-2 × MT serum stained structures resembling neurofibrillary tangles, neurites of neuritic plaques, and microglialike cells in SDAT brains, but no such staining pattern was detected in normal brain sections. In neurons isolated from SDAT brains, about 40% of the tangles were labeled by the anti-human-2×MT serum with an identical pattern. Other antiserums tested did not preferentially bind tanglelike structures in tissue sections and bound to less than 5% of the tangles in isolated neurons. These results suggest that the antigenic sites of tubulin and NF proteins are not shared by neurofibrillary tangles. Different from the calf preparation, the human-2 × MT fractions contained a prominent protein band that was identical to ferritin in molecular weight and cross-reacted with anti-human-2 × MT and anti-human-ferritin serums. However, antiserums to this ferritinlike protein, or anti-ferritin, did not stain neurofibrillary tangles. Although neither the calf 2 × MT nor two other human MT fractions failed to elicit an antiserum that stained tangles, these fractions were able to remove the antihuman-2 × MT serum activity that binds to tangles. The data suggest that the protein (or proteins) that makes up neurofibrillary tangles of SDAT is present in various quantities in microtubule fractions of normal brain. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6Figure 7Figure 8Figure 9Figure 10Figure 11Figure 12Figure 13Figure 14Figure 15Figure 16 PMID:7020426

A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat.

PubMed

Cheah, Joleen S; Yamada, Soichiro

2017-12-02

Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to traditional co-immunoprecipitation. In this protocol, a mutant biotin ligase promiscuously labels proximal binding partners with biotin, and resulting biotinylated proteins are purified using streptavidin conjugated beads. This approach does not require preservation of protein complexes in vitro, making it an ideal approach to identify transient or weak protein complexes. However, due to the high affinity bond between streptavidin and biotin, elution of biotinylated proteins from streptavidin conjugated beads requires harsh denaturing conditions, which are often incompatible with downstream processing. To effectively release biotinylated proteins bound to streptavidin conjugated beads, we designed a series of experiments to determine optimal binding and elution conditions. Interestingly, the concentrations of SDS and IGEPAL-CA630 during the incubation with streptavidin conjugated beads were the key to effective elution of biotinylated proteins using excess biotin and heating. This protocol provides an alternative method to isolate biotinylated proteins from streptavidin conjugated beads that is suitable for further downstream analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

PubMed

Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M

1992-02-01

The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs. The presence in shark liver of an FABP which differs substantially in primary structure from mammalian liver FABP, while being closely related to the FABP expressed in mammalian heart muscle, peripheral nerve myelin and adipocytes, opens a further dimension regarding the question of the existence of structure-dependent and tissue-specific specialization of FABP function in lipid metabolism.

A Herpesvirus Ribosome-Associated, RNA-Binding Protein Confers a Growth Advantage upon Mutants Deficient in a GADD34-Related Function†

PubMed Central

Mulvey, Matthew; Poppers, Jeremy; Ladd, Alison; Mohr, Ian

1999-01-01

The herpes simplex virus type 1 γ34.5 gene product and the cellular GADD34 protein both contain similar domains that can regulate the activity of eukaryotic initiation factor 2 (eIF2), a critical translation initiation factor. Viral mutants that lack the GADD34-related function grow poorly on a variety of malignant human cells, as activation of the cellular PKR kinase leads to the accumulation of inactive, phosphorylated eIF2 at late times postinfection. Termination of translation prior to the completion of the viral reproductive cycle leads to impaired growth. Extragenic suppressors that regain the ability to synthesize proteins efficiently in the absence of the viral GADD34-related function have been isolated. These suppressor alleles are dominant in trans and affect the steady-state accumulation of several viral mRNA species. We demonstrate that deregulated expression of Us11, a virus-encoded RNA-binding, ribosome-associated protein is necessary and sufficient to confer a growth advantage upon viral mutants that lack a GADD34-related function. Ectopic expression of Us11 reduces the accumulation of the activated cellular PKR kinase and allows for sustained protein synthesis. Thus, an RNA-binding, ribosome-associated protein (Us11) and a GADD34-related protein (γ34.5) both function in a signal pathway that regulates translation by modulating eIF2 phosphorylation. PMID:10074192