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Sample records for fibronectin binding motif

  1. A Novel Fibronectin Binding Motif in MSCRAMMs Targets F3 Modules

    PubMed Central

    Prabhakaran, Sabitha; Liang, Xiaowen; Skare, Jonathan T.; Potts, Jennifer R.; Höök, Magnus

    2009-01-01

    Background BBK32 is a surface expressed lipoprotein and fibronectin (Fn)-binding microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Borrelia burgdorferi, the causative agent of Lyme disease. Previous studies from our group showed that BBK32 is a virulence factor in experimental Lyme disease and located the Fn-binding region to residues 21–205 of the lipoprotein. Methodology/Principal Findings Studies aimed at identifying interacting sites between BBK32 and Fn revealed an interaction between the MSCRAMM and the Fn F3 modules. Further analysis of this interaction showed that BBK32 can cause the aggregation of human plasma Fn in a similar concentration-dependent manner to that of anastellin, the superfibronectin (sFn) inducing agent. The resulting Fn aggregates are conformationally distinct from plasma Fn as indicated by a change in available thermolysin cleavage sites. Recombinant BBK32 and anastellin affect the structure of Fn matrices formed by cultured fibroblasts and inhibit endothelial cell proliferation similarly. Within BBK32, we have located the sFn-forming activity to a region between residues 160 and 175 which contains two sequence motifs that are also found in anastellin. Synthetic peptides mimicking these motifs induce Fn aggregation, whereas a peptide with a scrambled sequence motif was inactive, suggesting that these motifs represent the sFn-inducing sequence. Conclusions/Significance We conclude that BBK32 induces the formation of Fn aggregates that are indistinguishable from those formed by anastellin. The results of this study provide evidence for how bacteria can target host proteins to manipulate host cell activities. PMID:19404402

  2. Interaction of N-terminal fragments of fibronectin with synthetic and recombinant D motifs from its binding protein on Staphylococcus aureus studied using fluorescence anisotropy.

    PubMed

    Huff, S; Matsuka, Y V; McGavin, M J; Ingham, K C

    1994-06-03

    The N-terminal 29-kDa fragment of fibronectin (Fn29K) contains five type I "finger" modules. It binds to heparin, fibrin, and bacteria and is involved in fibronectin (Fn) matrix assembly. Binding to Staphylococcus aureus involves a cell wall-associated protein that contains approximately three repeats of a 38-residue D motif (Signäs, C., Raucci, G., Jönsson, K., Lindgren, P.-E., Anantharamaiah, G.M., Höök, M., and Lindberg, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 699-703). Synthetic peptides representing D1, D2, and D3, when labeled with fluorescein isothiocyanate (FITC), exhibited increases in fluorescence anisotropy upon addition of Fn29K but not other Fn fragments. The response could be reversed by titration with unlabeled peptides to yield inhibition constants that agreed with the dissociation constants obtained by fitting the initial response. Values of Kd ranged between 2 and 12 microM, with D3 having the highest affinity. Specificity of D3 for Fn29K was further illustrated by the fact that its C-terminal half (D3b, Lys801 to Lys821), when immobilized, selectively adsorbed Fn29K from a thermolysin digest of fibronectin. The binding site in Fn was further localized within Fn29K by analyzing smaller proteolytic or recombinant subfragments. Those containing fingers, F3-5 and F4-5, were purified on D3b-Sepharose and bound FITC-D3b with Kd values of 4-6 microM. Subfragments containing pairs of fingers 1-2, 2-3, or single fingers 1, 4, or 5 were inactive. Whole D1-3, expressed in Escherichia coli and labeled with fluorescein, bound 1.9 mol/mol of Fn29K with Kd = 1.5 nM. F4-5 and F2-3 bound with respective Kd values of 0.35 and 4.4 microM. These and other results indicate that binding of the individual D region peptides is mediated through their C-terminal halves, primarily to fingers 4 and 5 of fibronectin. The possible basis of the much higher affinity of D1-3 is discussed.

  3. Binding of fibronectin to Staphylococcus strains.

    PubMed Central

    Switalski, L M; Rydén, C; Rubin, K; Ljungh, A; Höök, M; Wadström, T

    1983-01-01

    Fibronectin, a major protein component of plasma and loose connective tissue has previously been shown to bind to several strains of Staphylococcus aureus. We examined a large number of strains of different species of Staphylococcus with respect to their ability to bind fibronectin. The relative numbers of strains defined as fibronectin-binders among the different species were as follows: S. aureus (22 of 23), S. haemolyticus (5 of 5), S. warneri (8 of 11), S. hyicus (5 of 6), S. hominis (13 of 17), S. saprophyticus (11 of 20), S. epidermidis (4 of 7), and S. simulans (8 of 10). Only three species showed a predominance of nonbinders over binders: S. capitis (4 of 14), S. xylosus (0 of 4), and S. cohnii (3 of 11). These data indicate that staphylococcal species isolated from soft tissue infections frequently have the ability to bind fibronectin and suggest that the ability to bind to this protein may contribute to the virulence of coagulase-positive and coagulase-negative staphylococci. PMID:6315582

  4. Identification of a fibronectin-binding protein from Staphylococcus epidermidis.

    PubMed

    Williams, Rachel J; Henderson, Brian; Sharp, Lindsay J; Nair, Sean P

    2002-12-01

    Staphylococcus epidermidis has been reported to bind to a number of host cell extracellular matrix proteins, including fibronectin. Here we report the identification of a fibronectin-binding protein from S. epidermidis. A phage display library of S. epidermidis genomic DNA was constructed and panned against immobilized fibronectin. A number of phagemid clones containing overlapping inserts were identified, and one of these clones, pSE109FN, contained a 1.4-kb insert. Phage pSE109FN was found to bind to fibronectin but not to collagen, fibrinogen, laminin, or vitronectin. However, pSE109FN also bound to heparin, hyaluronate, and plasminogen, although to a lesser extent than it bound to fibronectin. Analysis of The Institute for Genomic Research S. epidermidis genome sequence database revealed a 1.85-kb region within a putative 30.5-kb open reading frame, to which the overlapping DNA inserts contained within the fibronectin-binding phagemids mapped. We have designated the gene encoding the fibronectin-binding domain embp. A recombinant protein, Embp32, which encompassed the fibronectin-binding domain of Embp, blocked the binding of S. epidermidis, but not the binding of Staphylococcus aureus, to fibronectin. In contrast, a recombinant protein, FnBPB[D1-D4], spanning the fibronectin-binding domain of the S. aureus fibronectin-binding protein FnBPB, blocked binding of S. aureus to fibronectin but had a negligible effect on the binding of S. epidermidis.

  5. Matrix Gla Protein Binds to Fibronectin and Enhances Cell Attachment and Spreading on Fibronectin

    PubMed Central

    Nishimoto, Satoru Ken; Nishimoto, Miyako

    2014-01-01

    Background. Matrix Gla protein (MGP) is a vitamin K-dependent, extracellular matrix protein. MGP is a calcification inhibitor of arteries and cartilage. However MGP is synthesized in many tissues and is especially enriched in embryonic tissues and in cancer cells. The presence of MGP in those instances does not correlate well with the calcification inhibitory role. This study explores a potential mechanism for MGP to bind to matrix proteins and alter cell matrix interactions. Methods. To determine whether MGP influences cell behavior through interaction with fibronectin, we studied MGP binding to fibronectin, the effect of MGP on fibronectin mediated cell attachment and spreading and immunolocalized MGP and fibronectin. Results. First, MGP binds to fibronectin. The binding site for MGP is in a specific fibronectin fragment, called III1-C or anastellin. The binding site for fibronectin is in a MGP C-terminal peptide comprising amino acids 61–77. Second, MGP enhances cell attachment and cell spreading on fibronectin. MGP alone does not promote cell adhesion. Third, MGP is present in fibronectin-rich regions of tissue sections. Conclusions. MGP binds to fibronectin. The presence of MGP increased cell-fibronectin interactions. PMID:25210519

  6. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  7. SdrI of Staphylococcus saprophyticus is a multifunctional protein: localization of the fibronectin-binding site.

    PubMed

    Sakinç, Türkân; Kleine, Britta; Michalski, Nadine; Kaase, Martin; Gatermann, Sören G

    2009-11-01

    Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, expresses the surface protein SdrI, a member of the serine-aspartate repeat (SD) protein family. Here we analyse the fibronectin-binding ability of SdrI, as S. saprophyticus is known to bind fibronectin and there is no known SD protein with this function. This protein does not contain the binding motif typical for fibronectin-binding proteins. Using recombinant fragments of SdrI, we localized the binding domain in the A region and show that SdrI bound to the N-terminal 30-kDa fragment of fibronectin. The fibronectin-binding function was shown in the natural host using an SdrI knockout mutant that showed decreased binding to fibronectin compared with wild-type strain 7108.

  8. Allosteric Regulation of Fibronectin/α5β1 Interaction by Fibronectin-Binding MSCRAMMs

    PubMed Central

    Liang, Xiaowen; Garcia, Brandon L.; Visai, Livia; Prabhakaran, Sabitha; Meenan, Nicola A. G.; Potts, Jennifer R.; Humphries, Martin J.; Höök, Magnus

    2016-01-01

    Adherence of microbes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with α5β1 integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/α5β1integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/α5β1 integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/α5β1 on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic α5β1 interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/α5β1 affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs. PMID:27434228

  9. 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.

  10. Fibronectin receptors of mononuclear phagocytes: Binding characteristics and biochemical isolation

    SciTech Connect

    Garcia-Pardo, A.; Ferreira, O.C.; Valinsky, J.; Bianco, C. )

    1989-04-01

    Fibronectin receptors on mononuclear phagocytes are involved in the localization of monocytes at inflammatory sites and in the subsequent expression of macrophage-like phenotypes. In this study, the authors have investigated the hypothesis that preteolytically derived fragments of fibronectin may interfere with binding of fibronectin to monocytes in the extracellular matrix. They report on the reactivity of U937 cells with an 80-kDa tryptic fragment of fibronectin which contains the cell-binding domain but lacks the gelatin/collagen-binding domain. U937 cells attached to surfaces coated with the 80-kDa fragment as well as with intact fibronectin. Preincubation of the cells with the 80-kDa fragment inhibited attachment to both surfaces while intact fibronectin had little or no inhibitory effect. This complex resolved into a single diffuse band of 144 kDa upon reduction. Binding of the protein complex to the affinity column required divalent cations. The complex bound to wheat germ agglutinin and could be specifically eluted by N-acetylglucosamine. Similar cell-surface proteins were isolated from peripheral blood monocytes.

  11. Crystal structure of a heparin- and integrin-binding segment of human fibronectin.

    PubMed Central

    Sharma, A; Askari, J A; Humphries, M J; Jones, E Y; Stuart, D I

    1999-01-01

    The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert. PMID:10075919

  12. The role of the fibronectin IGD motif in stimulating fibroblast migration.

    PubMed

    Millard, Christopher J; Ellis, Ian R; Pickford, Andrew R; Schor, Ana M; Schor, Seth L; Campbell, Iain D

    2007-12-07

    The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 10(6) times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN.

  13. Staphylococcus aureus fibronectin binding proteins A and B possess a second fibronectin binding region that may have biological relevance to bone tissues.

    PubMed

    Williams, R J; Henderson, B; Nair, S P

    2002-05-01

    Staphylococcus aureus is a major human pathogen that has a propensity for targeting to bone tissues and thereby causing bone disease. A plausible hypothesis is that S. aureus targets to bone using the MSCRAMM family of surface proteins possessed by this organism. Two such proteins that have recently been shown to be important in bone infections are the S. aureus fibronectin binding proteins (FnBP) A and B. To identify fibronectin-binding domains from S. aureus that have biological relevance to bone, a phage display library of S. aureus genomic DNA was constructed and panned sequentially against immobilized fibronectin and cultured osteoblasts. Using this system, phage displaying a second fibronectin-binding region within the N-terminal part of FnBPA and FnBPB, which is distinct from the primary fibronectin-binding domain located within the D repeat region of these proteins, was isolated. Phage displaying this second region bound to both immobilized fibronectin and to osteoblasts and/or the extracellular matrix synthesized by these cells, thereby suggesting a biological relevance for these regions in S. aureus binding to bone tissues. Analysis of these binding regions for their ability to bind to other extracellular matrix proteins revealed a preference for fibronectin, with slight binding to fibrinogen and no binding to collagen or laminin.

  14. An RNA motif that binds ATP

    NASA Technical Reports Server (NTRS)

    Sassanfar, M.; Szostak, J. W.

    1993-01-01

    RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

  15. Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin

    PubMed Central

    1993-01-01

    The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin- binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix. PMID:8468356

  16. Dissociation Rate Constants of Human Fibronectin Binding to Fibronectin-binding Proteins on Living Staphylococcus aureus Isolated from Clinical Patients*

    PubMed Central

    Casillas-Ituarte, Nadia N.; Lower, Brian H.; Lamlertthon, Supaporn; Fowler, Vance G.; Lower, Steven K.

    2012-01-01

    Staphylococcus aureus is part of the indigenous microbiota of humans. Sometimes, S. aureus bacteria enter the bloodstream, where they form infections on implanted cardiovascular devices. A critical, first step in such infections is a bond that forms between fibronectin-binding protein (FnBP) on S. aureus and host proteins, such as fibronectin (Fn), that coat the surface of implants in vivo. In this study, native FnBPs on living S. aureus were shown to form a mechanically strong conformational structure with Fn by atomic force microscopy. The tensile acuity of this bond was probed for 46 bloodstream isolates, each from a patient with a cardiovascular implant. By analyzing the force spectra with the worm-like chain model, we determined that the binding events were consistent with a multivalent, cluster bond consisting of ∼10 or ∼80 proteins in parallel. The dissociation rate constant (koff, s−1) of each multibond complex was determined by measuring strength as a function of the loading rate, normalized by the number of bonds. The bond lifetime (1/koff) was two times longer for bloodstream isolates from patients with an infected device (1.79 or 69.47 s for the 10- or 80-bond clusters, respectively; n = 26 isolates) relative to those from patients with an uninfected device (0.96 or 34.02 s; n = 20 isolates). This distinction could not be explained by different amounts of FnBP, as confirmed by Western blots. Rather, amino acid polymorphisms within the Fn-binding repeats of FnBPA explain, at least partially, the statistically (p < 0.05) longer bond lifetime for isolates associated with an infected cardiovascular device. PMID:22219202

  17. Proteolytically stabilizing fibronectin without compromising cell and gelatin binding activity.

    PubMed

    Zhang, Chen; Ramanathan, Anand; Karuri, Nancy Wangechi

    2015-01-01

    Excessive proteolytic degradation of fibronectin (FN) has been implicated in impaired tissue repair in chronic wounds. We previously reported two strategies for stabilizing FN against proteolytic degradation; the first conjugated polyethylene glycol (PEG) through cysteine residues and the second conjugated PEG chains of varying molecular weight on lysine residues. PEGylation of FN via lysine residues resulted in increased resistance to proteolysis with increasing PEG size, but an overall decrease in biological activity, as characterized by cell and gelatin binding. Our latest method to stabilize FN against proteolysis masks functional regions in the protein during lysine PEGylation. FN is PEGylated while it is bound to gelatin Sepharose beads with 2, 5, and 10 kDa PEG precursors. This results in partially PEGylated FN that is more stable than native FN and whose proteolytic stability increases with PEG molecular weight. Unlike completely PEGylated FN, partially PEGylated FN has cell adhesion, gelatin binding, and matrix assembly responses that are comparable to native FN. This is new evidence of how PEGylation variables can be used to stabilize FN while retaining its activity. The conjugates developed herein can be used to dissect molecular mechanisms mediated by FN stability and functionality, and address the problem of FN degradation in chronic wounds.

  18. Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation.

    PubMed

    Casillas-Ituarte, Nadia N; Cruz, Carlos H B; Lins, Roberto D; DiBartola, Alex C; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F T; Sierra-Hernández, M Roxana; Lower, Steven K

    2017-04-11

    The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous single nucleotide polymorphisms (SNPs) in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight, 20-mer peptide variants containing amino acids A, H, I, K, N, and Q at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 (FnBR-9) of FnBPA. Experimentally measured bond lifetimes (1/koff ) and dissociation constants (Kd = koff / kon ), determined by mechanically dissociating the Fn-peptide complex at loading rates relevant to the cardiovascular system varied from the lowest-affinity H782A+K786A peptide (0.011 sec, 747 µM) to the highest-affinity H782Q+K786N peptide (0.192 sec, 15.7 µM). These AFM results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q+K786I (Kd(app) = 0.2 to 0.5 µM. as determined by SPR) compared with the lowest-affinity double alanine peptide (Kd(app) = 3.8 µM). Together, these findings demonstrate that amino acid substitutions in FnBR-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA) among clinical isolates of S. aureus that cause endovascular infections.

  19. Binding of human fibronectin to group A, C, and G streptococci.

    PubMed Central

    Myhre, E B; Kuusela, P

    1983-01-01

    A total of 387 bacterial strains belonging to 35 species were tested in direct binding experiments for the uptake of purified radiolabeled human fibronectin. Positive binding was found in group A, C, and G streptococci and in Staphylococcus aureus. The group C streptococcal species Streptococcus equisimilis, Streptococcus zooepidemicus, Streptococcus equi, and Streptococcus dysgalactiae were uniformly reactive with fibronectin. Beta-hemolytic bovine group G streptococci showed the same degree of reactivity as human group G strains. In contrast, only 4 out of 15 alpha-hemolytic bovine group G strains were able to bind fibronectin. The uptake of fibronectin measured at room temperature with a human group G streptococcus was a slow, time-dependent process with maximum binding after approximately 1 h. Human polyclonal immunoglobulin G and serum albumin tested in inhibition experiments did not affect the fibronectin binding. Fibronectin seems, therefore, to interact with a surface component that is different from the specific binding sites previously described for human immunoglobulin G and serum albumin. PMID:6299959

  20. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

    PubMed Central

    Talay, S R; Valentin-Weigand, P; Jerlström, P G; Timmis, K N; Chhatwal, G S

    1992-01-01

    The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to

  1. Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M

    PubMed Central

    AFSHAR, Davoud; POURMAND, Mohammad Reza; JEDDI-TEHRANI, Mahmood; SABOOR YARAGHI, Ali Akbar; AZARSA, Mohammad; SHOKRI, Fazel

    2016-01-01

    Background: Choline-binding proteins (CBPs) are a group of surface-exposed proteins, which play crucial and physiological roles in Streptococcus pneumoniae. The novel member of CBPs, choline-binding protein M (CbpM) may have binding activity to plasma proteins. This study aimed to clone and express CbpM and demonstrate its interaction with plasma proteins and patients’ sera. Methods: The total length of cbpM gene was cloned in pET21a vector and expressed in BL21 expression host. Verification of recombinant protein was evaluated by Western blot using anti-His tag monoclonal antibody. Binding ability of the recombinant protein to plasma proteins and the interaction with patients’ sera were assessed by Western blot and ELISA methods. Results: The cbpM gene was successfully cloned into pET21a and expressed in BL21 host. Binding activity to fibronectin and fibrinogen and antibody reaction of CbpM to patients’ sera was demonstrated by Western blot and ELISA methods, respectively. Conclusion: CbpM is one of the pneumococcal surface-exposed proteins, which mediates pneumococcal binding to fibronectin and fibrinogen proteins. PMID:28053927

  2. Identification and characterization of a fibronectin-binding protein from Granulicatella adiacens.

    PubMed

    Yamaguchi, T; Soutome, S; Oho, T

    2011-12-01

    The interaction of microorganisms with fibronectin plays an important role in infective endocarditis. Granulicatella adiacens is a member of the oral microbiota, formerly known as nutritionally variant streptococci, and is often isolated from endocarditis patients. In the present study we identified a surface protein, designated Cha, which binds to fibronectin, by a plaque hybridization procedure using the cshA sequence as probe, which encodes a fibronectin-binding molecule of Streptococcus gordonii DL1. The cha sequence was highly homologous to cshA and encoded a product of 2351 amino acid residues. The protein comprised a unique sequence in the N-terminal half region. The C-terminal region contained nine complete, and one incomplete, 115-amino acid residue repeat blocks. Among eight strains of nutritionally variant streptococci, three G. adiacens strains and one Abiotrophia defectiva strain carried the cha gene. Heterologous expression studies suggested that Cha adhered to immobilized fibronectin, and that this function was located in the unique region. Recombinant Cha protein also adhered to immobilize fibronectin and partially inhibited adherence of G. adiacens to fibronectin in a dose-dependent manner. These results suggest that Cha is a cell surface protein that mediates adherence of G. adiacens to fibronectin.

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

    SciTech Connect

    Lower, Steven; Lamlertthon, Supaporn; Casillas-Ituarte, Nadia; Lins, Roberto D.; Yongsunthon, Ruchirej; Taylor, Eric S.; DiBartola, Alex; Edmondson, Catherine; McIntyre, Lauren M.; Reller, L. Barth; Que, Yok-Ai; Ros, Robert; Lower, Brian; Fowler, Vance

    2011-11-08

    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 from 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.

  4. A survey of motif finding Web tools for detecting binding site motifs in ChIP-Seq data.

    PubMed

    Tran, Ngoc Tam L; Huang, Chun-Hsi

    2014-02-20

    ChIP-Seq (chromatin immunoprecipitation sequencing) has provided the advantage for finding motifs as ChIP-Seq experiments narrow down the motif finding to binding site locations. Recent motif finding tools facilitate the motif detection by providing user-friendly Web interface. In this work, we reviewed nine motif finding Web tools that are capable for detecting binding site motifs in ChIP-Seq data. We showed each motif finding Web tool has its own advantages for detecting motifs that other tools may not discover. We recommended the users to use multiple motif finding Web tools that implement different algorithms for obtaining significant motifs, overlapping resemble motifs, and non-overlapping motifs. Finally, we provided our suggestions for future development of motif finding Web tool that better assists researchers for finding motifs in ChIP-Seq data.

  5. Definition of the native and denatured type II collagen binding site for fibronectin using a recombinant collagen system.

    PubMed

    An, Bo; Abbonante, Vittorio; Yigit, Sezin; Balduini, Alessandra; Kaplan, David L; Brodsky, Barbara

    2014-02-21

    Interaction of collagen with fibronectin is important for extracellular matrix assembly and regulation of cellular processes. A fibronectin-binding region in collagen was identified using unfolded fragments, but it is not clear if the native protein binds fibronectin with the same primary sequence. A recombinant bacterial collagen is utilized to characterize the sequence requirement for fibronectin binding. Chimeric collagens were generated by inserting the putative fibronectin-binding region from human collagen into the bacterial collagen sequence. Insertion of a sufficient length of human sequence conferred fibronectin affinity. The minimum sequence requirement was identified as a 6-triplet sequence near the unique collagenase cleavage site and was the same in both triple-helix and denatured states. Denaturation of the chimeric collagen increased its affinity for fibronectin, as seen for mammalian collagens. The fibronectin binding recombinant collagen did not contain hydroxyproline, indicating hydroxyproline is not essential for binding. However, its absence may account, in part, for the higher affinity of the native chimeric protein and the lower affinity of the denatured protein compared with type II collagen. Megakaryocytes cultured on chimeric collagen with fibronectin affinity showed improved adhesion and differentiation, suggesting a strategy for generating bioactive materials in biomedical applications.

  6. Antibody Response to Fibronectin-Binding Adhesin FnbpA in Patients with Staphylococcus aureus Infections

    PubMed Central

    Casolini, Fabrizia; Visai, Livia; Joh, Danny; Conaldi, Pier Giulio; Toniolo, Antonio; Höök, Magnus; Speziale, Pietro

    1998-01-01

    We have analyzed antibody reactivity to a fibronectin-binding microbial surface component that recognizes adhesive matrix molecules (MSCRAMM) in blood plasma collected from patients with staphylococcal infections. All patients had elevated levels of anti-MSCRAMM antibodies compared to those of young children who, presumably, had not been exposed to staphylococcal infections. The anti-MSCRAMM antibodies preferentially reacted with the ligand-binding repeat domain of the adhesin. However, these antibodies did not inhibit fibronectin binding. Essentially, all patients had antibodies which specifically recognized the fibronectin-MSCRAMM complex but not the isolated components. Epitopes recognized by these anti-ligand-induced binding sites antibodies were found in each repeat unit of the MSCRAMM. These results demonstrate that staphylococci have bound fibronectin some time during infection and that each repeat unit in the MSCRAMM can engage in ligand binding. Furthermore, our previously proposed model, suggesting that an unordered structure in the MSCRAMM undergoes a conformational change upon ligand binding (K. House-Pompeo, Y. Xu, D. Joh, P. Speziale, and M. Höök, J. Biol. Chem. 271:1379–1384, 1996), is presumably operational in patients during infections. PMID:9784554

  7. Thrombomodulin promotes focal adhesion kinase activation and contributes to angiogenesis by binding to fibronectin

    PubMed Central

    Hsu, Yun-Yan; Shi, Guey-Yueh; Wang, Kuan-Chieh; Ma, Chih-Yuan; Cheng, Tsung-Lin; Wu, Hua-Lin

    2016-01-01

    Angiogenesis promotes tumor growth and metastasis. Cell adhesion molecules interact with the extracellular matrix (ECM) and increase cell adhesion and migration during angiogenesis. Thrombomodulin (TM) is a cell surface transmembrane glycoprotein expressed in endothelial cells. However, the function and significance of TM in cell-matrix interactions and angiogenesis remain unclear. Here, we first demonstrated that recombinant lectin-like domain of TM interacts with an ECM protein, fibronectin, and identified the N-terminal 70-kDa domain of fibronectin as the TM-binding site. Exogenous expression of TM in TM-deficient A2058 melanoma cells enhanced cell adhesion and migration on fibronectin and invasion on Matrigel. In addition, TM increased focal adhesion kinase (FAK) phosphorylation and matrix metalloproteinase-9 production. In mice bearing subcutaneous B16F10 melanoma tumors, immunofluorescence analysis indicated that TM was highly expressed and co-localized with fibronectin on the tumor vasculature. The interaction between TM and fibronectin in tumor blood vessels was also validated by the proximity ligation assay. In human umbilical vein endothelial cells, up-regulation of TM by vascular endothelial growth factor (VEGF), a tumor angiogenic factor, promoted cell adhesion and tube formation, whereas TM knockdown by RNA interference attenuated VEGF-induced cell adhesion and tube formation. In summary, TM promotes angiogenesis by enhancing cell adhesion, migration, and FAK activation through interaction with fibronectin. TM may represent a novel target for inhibiting tumor angiogenesis. PMID:27602495

  8. Adhesive surface proteins of Erysipelothrix rhusiopathiae bind to polystyrene, fibronectin, and type I and IV collagens.

    PubMed

    Shimoji, Yoshihiro; Ogawa, Yohsuke; Osaki, Makoto; Kabeya, Hidenori; Maruyama, Soichi; Mikami, Takeshi; Sekizaki, Tsutomu

    2003-05-01

    Erysipelothrix rhusiopathiae is a gram-positive bacterium that causes erysipelas in animals and erysipeloid in humans. We found two adhesive surface proteins of E. rhusiopathiae and determined the nucleotide sequences of the genes, which were colocalized and designated rspA and rspB. The two genes were present in all of the serovars of E. rhusiopathiae strains examined. The deduced RspA and RspB proteins contain the C-terminal anchoring motif, LPXTG, which is preceded by repeats of consensus amino acid sequences. The consensus sequences are composed of 78 to 92 amino acids and repeat 16 and 3 times in RspA and RspB, respectively. Adhesive surface proteins of other gram-positive bacteria, including Listeria monocytogenes adhesin-like protein, Streptococcus pyogenes protein F2 and F2-like protein, Streptococcus dysgalactiae FnBB, and Staphylococcus aureus Cna, share the same consensus repeats. Furthermore, the N-terminal regions of RspA and RspB showed characteristics of the collagen-binding domain that was described for Cna. RspA and RspB were expressed in Escherichia coli as histidine-tagged fusion proteins and purified. The recombinant proteins showed a high degree of capacity to bind to polystyrene and inhibited the binding of E. rhusiopathiae onto the abiotic surface in a dose dependent manner. In a solid-phase binding assay, both of the recombinant proteins bound to fibronectin, type I and IV collagens, indicating broad spectrum of their binding ability. It was suggested that both RspA and RspB were exposed on the cell surface of E. rhusiopathiae, as were the bacterial cells agglutinated by the anti-RspA immunoglobulin G (IgG) and anti-RspB IgG. RspA and RspB were present both in surface-antigen extracts and the culture supernatants of E. rhusiopathiae Fujisawa-SmR (serovar 1a) and SE-9 (serovar 2). The recombinant RspA, but not RspB, elicited protection in mice against experimental challenge. These results suggest that RspA and RspB participate in initiation

  9. The evolution of tenascins and fibronectin.

    PubMed

    Adams, Josephine C; Chiquet-Ehrismann, Ruth; Tucker, Richard P

    2015-01-01

    Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes.

  10. The evolution of tenascins and fibronectin

    PubMed Central

    Adams, Josephine C; Chiquet-Ehrismann, Ruth; Tucker, Richard P

    2015-01-01

    Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes. PMID:25482621

  11. Endovascular Infections Caused by Methicillin-Resistant Staphylococcus aureus Are Linked to Clonal Complex-Specific Alterations in Binding and Invasion Domains of Fibronectin-Binding Protein A as Well as the Occurrence of fnbB

    PubMed Central

    Xiong, Yan Q.; Sharma-Kuinkel, Batu K.; Casillas-Ituarte, Nadia N.; Fowler, Vance G.; Rude, Thomas; DiBartola, Alex C.; Lins, Roberto D.; Abdel-Hady, Wessam; Bayer, Arnold S.

    2015-01-01

    Endovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates (n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains (n = 5) versus CC45 strains (n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin (P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA, which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB, particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and

  12. Endovascular infections caused by methicillin-resistant Staphylococcus aureus are linked to clonal complex-specific alterations in binding and invasion domains of fibronectin-binding protein A as well as the occurrence of fnbB.

    PubMed

    Xiong, Yan Q; Sharma-Kuinkel, Batu K; Casillas-Ituarte, Nadia N; Fowler, Vance G; Rude, Thomas; DiBartola, Alex C; Lins, Roberto D; Abdel-Hady, Wessam; Lower, Steven K; Bayer, Arnold S

    2015-12-01

    Endovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates (n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains (n = 5) versus CC45 strains (n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin (P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA, which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB, particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and

  13. Crystal structures of fibronectin-binding sites from Staphylococcus aureus FnBPA in complex with fibronectin domains

    PubMed Central

    Bingham, Richard J.; Rudiño-Piñera, Enrique; Meenan, Nicola A. G.; Schwarz-Linek, Ulrich; Turkenburg, Johan P.; Höök, Magnus; Garman, Elspeth F.; Potts, Jennifer R.

    2008-01-01

    Staphylococcus aureus can adhere to and invade endothelial cells by binding to the human protein fibronectin (Fn). FnBPA and FnBPB, cell wall-attached proteins from S. aureus, have multiple, intrinsically disordered, high-affinity binding repeats (FnBRs) for Fn. Here, 30 years after the first report of S. aureus/Fn interactions, we present four crystal structures that together comprise the structures of two complete FnBRs, each in complex with four of the N-terminal modules of Fn. Each ≈40-residue FnBR forms antiparallel strands along the triple-stranded β-sheets of four sequential F1 modules (2–5F1) with each FnBR/2–5F1 interface burying a total surface area of ≈4,300 Å2. The structures reveal the roles of residues conserved between S. aureus and Streptococcus pyogenes FnBRs and show that there are few linker residues between FnBRs. The ability to form large intermolecular interfaces with relatively few residues has been proposed to be a feature of disordered proteins, and S. aureus/Fn interactions provide an unusual illustration of this efficiency. PMID:18713862

  14. Identification of Surface Proteins from Lactobacillus casei BL23 Able to Bind Fibronectin and Collagen.

    PubMed

    Muñoz-Provencio, Diego; Pérez-Martínez, Gaspar; Monedero, Vicente

    2011-03-01

    Strains of lactobacilli show the capacity to attach to extracellular matrix proteins. Cell-wall fractions of Lactobacillus casei BL23 enriched in fibronectin, and collagen-binding proteins were isolated. Mass spectrometry analysis of their protein content revealed the presence of stress-related proteins (GroEL, ClpL), translational elongation factors (EF-Tu, EF-G), oligopeptide solute-binding proteins, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The latter two enzymes were expressed in Escherichia coli and purified as glutathione-S-transferase (GST) fusion proteins, and their in vitro binding activity to fibronectin and collagen was confirmed. These results reinforce the idea that lactobacilli display on their surfaces a variety of moonlighting proteins that can be important in their adaptation to survive at intestinal mucosal sites and in the interaction with host cells.

  15. Structural and dynamical characterization of a biologically active unfolded fibronectin-binding protein from Staphylococcus aureus.

    PubMed

    Penkett, C J; Redfield, C; Jones, J A; Dodd, I; Hubbard, J; Smith, R A; Smith, L J; Dobson, C M

    1998-12-01

    A 130-residue fragment (D1-D4) taken from a fibronectin-binding protein of Staphylococcus aureus, which contains four fibronectin-binding repeats and is unfolded but biologically active at neutral pH, has been studied extensively by NMR spectroscopy. Using heteronuclear multidimensional techniques, the conformational properties of D1-D4 have been defined at both a global and a local level. Diffusion studies give an average effective radius of 26.2 +/- 0.1 A, approximately 75% larger than that expected for a globular protein of this size. Analysis of chemical shift, 3JHNalpha coupling constant, and NOE data show that the experimental parameters agree well overall with values measured in short model peptides and with predictions from a statistical model for a random coil. Sequences where specific features give deviations from these predictions for a random coil have however been identified. These arise from clustering of hydrophobic side chains and electrostatic interactions between charged groups. 15N relaxation studies demonstrate that local fluctuations of the chain are the dominant motional process that gives rise to relaxation of the 15N nuclei, with a persistence length of approximately 7-10 residues for the segmental motion. The consequences of the structural and dynamical properties of this unfolded protein for its biological role of binding to fibronectin have been considered. It is found that the regions of the sequence involved in binding have a high propensity for populating extended conformations, a feature that would allow a number of both charged and hydrophobic groups to be presented to fibronectin for highly specific binding.

  16. Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response

    PubMed Central

    1989-01-01

    Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125- fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex. PMID:2477381

  17. Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM

    PubMed Central

    Hymes, Jeffrey P.; Johnson, Brant R.; Barrangou, Rodolphe

    2016-01-01

    Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion of fbpB lost the ability to adhere to mucin and fibronectin in vitro. Homologues of fbpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside the L. acidophilus homology group. PMID:26921419

  18. Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM.

    PubMed

    Hymes, Jeffrey P; Johnson, Brant R; Barrangou, Rodolphe; Klaenhammer, Todd R

    2016-05-01

    Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion off bpB lost the ability to adhere to mucin and fibronectin in vitro Homologues off bpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside theL. acidophilus homology group.

  19. A Processed Multidomain Mycoplasma hyopneumoniae Adhesin Binds Fibronectin, Plasminogen, and Swine Respiratory Cilia

    PubMed Central

    Seymour, Lisa M.; Deutscher, Ania T.; Jenkins, Cheryl; Kuit, Tracey A.; Falconer, Linda; Minion, F. Chris; Crossett, Ben; Padula, Matthew; Dixon, Nicholas E.; Djordjevic, Steven P.; Walker, Mark J.

    2010-01-01

    Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (KD 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (KD 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae. PMID:20813843

  20. A processed multidomain mycoplasma hyopneumoniae adhesin binds fibronectin, plasminogen, and swine respiratory cilia.

    PubMed

    Seymour, Lisa M; Deutscher, Ania T; Jenkins, Cheryl; Kuit, Tracey A; Falconer, Linda; Minion, F Chris; Crossett, Ben; Padula, Matthew; Dixon, Nicholas E; Djordjevic, Steven P; Walker, Mark J

    2010-10-29

    Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (K(D) 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (K(D) 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae.

  1. Development and evaluation of a new kinetic assay for the quantitation of fibronectin gelatin-binding activity.

    PubMed

    Gelder, F B; Brown, S T; Moore, C A

    1985-10-01

    A new rapid and sensitive kinetic assay that measures the gelatin-binding activity of fibronectin has been developed. This assay is based on the rate of fibronectin-mediated aggregation of covalently coupled latex-gelatin particles. The addition of human plasma and serum resulted in aggregation rates proportional to the concentration of fibronectin in the test sample. This assay was inhibited by the addition of gelatin, demonstrating substrate specificity. This new assay requires 50 microliter of sample and can be performed within 5 minutes. Particle aggregation rates were affected by pH, heparin, and coupled gelatin concentration per milligram of latex. Maximum aggregation rates were observed at pH 8.0. Heparin was not an absolute requirement for particle aggregation but enhanced rates up to 1 U heparin/ml with little additive effect at greater concentrations. Heparin had a relatively greater effect on assays performed in acidic buffers. The concentration of gelatin per milligram of latex was rate limiting up to approximately 50 micrograms gelatin/mg latex with little change in aggregation rates at higher concentrations. Good correlation between total antigenic fibronectin (electroimmunoassay) and fibronectin gelatin-binding activity (latex-gelatin kinetic aggregation assay) was demonstrated in plasma from normal blood donors. This new assay will allow further definition of the relationship between fibronectin gelatin-binding activity and antigenic fibronectin in normal and pathophysiologic states.

  2. Mechanism of Fibronectin Binding to Human Trabecular Meshwork Exosomes and Its Modulation by Dexamethasone

    PubMed Central

    Klingeborn, Mikael; Stamer, W. Daniel

    2016-01-01

    Exosomes are emerging as important mediators of cell-matrix interactions by means of specific adhesion proteins. Changes in the tissue-specific exosomal protein expression may underlie pathological conditions whereby extracellular matrix turnover and homeostasis is disrupted. Ocular hypertension due to extracellular matrix accumulation in the trabecular meshwork is a hallmark of glucocorticoid-induced glaucoma. In the trabecular meshwork, exosomal fibronectin mediates cell matrix interactions at cellular structures called “invadosomes”. Trabecular meshwork cells use invadosomes to turn over their surrounding matrix and maintain passageways for flow of aqueous humor. In this study, we observed that human trabecular meshwork explants treated with dexamethasone released exosomes with significantly reduced amounts of fibronectin bound per exosome. Further, we found that exosome-fibronectin binding is heparan sulfate-dependent, consistent with our observation that trabecular meshwork exosomes are enriched in the heparin/heparan sulfate binding annexins A2 and A6. In this way, dexamethasone-treated explants released exosomes with a significant reduction in annexin A2 and A6 per exosome. Interestingly, we did not detect exosomal matrix metalloproteinases, but we identified abundant dipeptidyl peptidase 4, a serine protease whose activity was reduced on exosomes isolated from dexamethasone-treated explants. Together, our findings demonstrate mechanistically how corticosteroid-induced alterations in exosomal adhesion cargo and properties can account for the pathological matrix accumulation seen in many glaucoma patients. PMID:27783649

  3. A survey of motif finding Web tools for detecting binding site motifs in ChIP-Seq data

    PubMed Central

    2014-01-01

    Abstract ChIP-Seq (chromatin immunoprecipitation sequencing) has provided the advantage for finding motifs as ChIP-Seq experiments narrow down the motif finding to binding site locations. Recent motif finding tools facilitate the motif detection by providing user-friendly Web interface. In this work, we reviewed nine motif finding Web tools that are capable for detecting binding site motifs in ChIP-Seq data. We showed each motif finding Web tool has its own advantages for detecting motifs that other tools may not discover. We recommended the users to use multiple motif finding Web tools that implement different algorithms for obtaining significant motifs, overlapping resemble motifs, and non-overlapping motifs. Finally, we provided our suggestions for future development of motif finding Web tool that better assists researchers for finding motifs in ChIP-Seq data. Reviewers This article was reviewed by Prof. Sandor Pongor, Dr. Yuriy Gusev, and Dr. Shyam Prabhakar (nominated by Prof. Limsoon Wong). PMID:24555784

  4. Mapping the heparin-binding site on the 13-14F3 fragment of fibronectin.

    PubMed

    Sachchidanand; Lequin, Olivier; Staunton, David; Mulloy, Barbara; Forster, Mark J; Yoshida, Keiichi; Campbell, Iain D

    2002-12-27

    Fibronectin, a multifunctional glycoprotein of the extracellular matrix, plays a major role in cell adhesion. Various studies have revealed that the human 13th and 14th fibronectin type III domains (labeled (13)F3 and (14)F3 here) contain a heparin-binding site. Mapping of the heparin-binding sites of (13-14)F3, (13)F3, and (14)F3 by NMR chemical shift perturbation, isothermal titration calorimetry, and molecular modeling show that (13)F3 provides the dominant heparin-binding site and that the residues involved are within the first 29 amino acids of (13)F3. Predictions from earlier biochemical and modeling studies as well as the x-ray structure of (12-14)F3 were tested. It was shown that the positively charged residues that project into the solvent from the ABE face of the triple-stranded beta sheet on (13)F3 are involved in binding, but (14)F3 does not appear to contribute significantly to heparin binding.

  5. Fibronectin inhibits cytokine production induced by CpG DNA in macrophages without direct binding to DNA.

    PubMed

    Yoshida, Hiroyuki; Nishikawa, Makiya; Yasuda, Sachiyo; Toyota, Hiroyasu; Kiyota, Tsuyoshi; Takahashi, Yuki; Takakura, Yoshinobu

    2012-10-01

    Fibronectin (FN) is known to have four DNA-binding domains although their physiological significance is unknown. Primary murine peritoneal macrophages have been shown to exhibit markedly lower responsiveness to CpG motif-replete plasmid DNA (pDNA), Toll-like receptor-9 (TLR9) ligand, compared with murine macrophage-like cell lines. The present study was conducted to examine whether FN having DNA-binding domains is involved in this phenomenon. The expression of FN was significantly higher in primary macrophages than in a macrophage-like cell line, RAW264.7, suggesting that abundant FN might suppress the responsiveness in the primary macrophages. However, electrophoretic analysis revealed that FN did not bind to pDNA in the presence of a physiological concentration of divalent cations. Surprisingly, marked tumor necrosis factor - (TNF-)α production from murine macrophages upon CpG DNA stimulation was significantly reduced by exogenously added FN in a concentration-dependent manner but not by BSA, laminin or collagen. FN did not affect apparent pDNA uptake by the cells. Moreover, FN reduced TNF-α production induced by polyI:C (TLR3 ligand), and imiquimod (TLR7 ligand), but not by LPS (TLR4 ligand), or a non-CpG pDNA/cationic liposome complex. The confocal microscopic study showed that pDNA was co-localized with FN in the same intracellular compartment in RAW264.7, suggesting that FN inhibits cytokine signal transduction in the endosomal/lysosomal compartment. Taken together, the results of the present study has revealed, for the first time, a novel effect of FN whereby the glycoprotein modulates cytokine signal transduction via CpG-DNA/TLR9 interaction in macrophages without direct binding to DNA through its putative DNA-binding domains.

  6. Staphylococcus intermedius binding to immobilized fibrinogen, fibronectin and cytokeratin in vitro.

    PubMed

    Schmidt, Vanessa; Nuttall, Tim; Fazakerley, Jennie; McEwan, Neil

    2009-10-01

    Bacterial adhesion is a key step in colonization of the skin. Staphylococcus intermedius adheres strongly to canine and feline corneocytes, and adhesion is greater to corneocytes from dogs affected with atopic dermatitis, but comparatively little is known about adhesion-receptor interaction compared to S. aureus. The aim of this study was to compare the binding of S. intermedius isolates from healthy (n = 21) and atopic dogs (n = 33) to immobilized human fibronectin and epidermal cytokeratin and canine fibrinogen in vitro. Staphylococcus intermedius and the positive control S. aureus P1 exhibited concentration-dependent binding to all three protein layers. The negative control S. aureus Newman strain and S. hominis did not bind. The majority of S. intermedius isolates adhered strongly, and there was no significant difference between isolates from atopic and healthy dogs or from lesional or nonlesional skin of atopic dogs (fibronectin P = 0.971 and 0.837; fibrinogen P = 0.811 and 0.564; cytokeratin P = 0.409 and 0.564). These results suggest that S. intermedius may possess specific microbial components recognizing adhesive matrix molecules, like S. aureus, that bind to the substrates used in this study. Adherence and therefore colonization and infection in canine atopic dermatitis, however, are more likely to be related to host factors rather than the possession of specific virulence factors.

  7. Structure and ubiquitin binding of the ubiquitin-interacting motif

    SciTech Connect

    Fisher,R.; Wang, B.; Alam, S.; Higginson, D.; Robinson, H.; Sundquist, C.; Hill, C.

    2003-01-01

    Ubiquitylation is used to target proteins into a large number of different biological processes including proteasomal degradation, endocytosis, virus budding, and vacuolar protein sorting (Vps). Ubiquitylated proteins are typically recognized using one of several different conserved ubiquitin binding modules. Here, we report the crystal structure and ubiquitin binding properties of one such module, the ubiquitin-interacting motif (UIM). We found that UIM peptides from several proteins involved in endocytosis and vacuolar protein sorting including Hrs, Vps27p, Stam1, and Eps15 bound specifically, but with modest affinity (K{sub d} = 0.1-1 mM), to free ubiquitin. Full affinity ubiquitin binding required the presence of conserved acidic patches at the N and C terminus of the UIM, as well as highly conserved central alanine and serine residues. NMR chemical shift perturbation mapping experiments demonstrated that all of these UIM peptides bind to the I44 surface of ubiquitin. The 1.45 {angstrom} resolution crystal structure of the second yeast Vps27p UIM (Vps27p-2) revealed that the ubiquitin-interacting motif forms an amphipathic helix. Although Vps27p-2 is monomeric in solution, the motif unexpectedly crystallized as an antiparallel four-helix bundle, and the potential biological implications of UIM oligomerization are therefore discussed.

  8. Outer membrane protein DsrA is the major fibronectin-binding determinant of Haemophilus ducreyi.

    PubMed

    Leduc, Isabelle; White, C Dinitra; Nepluev, Igor; Throm, Robert E; Spinola, Stanley M; Elkins, Christopher

    2008-04-01

    The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains.

  9. Borrelia burgdorferi protein BBK32 binds to soluble fibronectin via the N-terminal 70-kDa region, causing fibronectin to undergo conformational extension.

    PubMed

    Harris, Gemma; Ma, Wenjiang; Maurer, Lisa M; Potts, Jennifer R; Mosher, Deane F

    2014-08-08

    BBK32 is a fibronectin (FN)-binding protein expressed on the cell surface of Borrelia burgdorferi, the causative agent of Lyme disease. There is conflicting information about where and how BBK32 interacts with FN. We have characterized interactions of a recombinant 86-mer polypeptide, "Bbk32," comprising the unstructured FN-binding region of BBK32. Competitive enzyme-linked assays utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies showed that Bbk32 binding involves both the fibrin-binding and the gelatin-binding domains of the 70-kDa N-terminal region (FN70K). Crystallographic and NMR analyses of smaller Bbk32 peptides complexed, respectively, with (2-3)FNI and (8-9)FNI, demonstrated that binding occurs by β-strand addition. Isothermal titration calorimetry indicated that Bbk32 binds to isolated FN70K more tightly than to intact FN. In a competitive enzyme-linked binding assay, complex formation with Bbk32 enhanced binding of FN with mAbIII-10 to the (10)FNIII module. Thus, Bbk32 binds to multiple FN type 1 modules of the FN70K region by a tandem β-zipper mechanism, and in doing so increases accessibility of FNIII modules that interact with other ligands. The similarity in the FN-binding mechanism of BBK32 and previously studied streptococcal proteins suggests that the binding and associated conformational change of FN play a role in infection.

  10. Decreased RNA-binding motif 5 expression is associated with tumor progression in gastric cancer.

    PubMed

    Kobayashi, Takahiko; Ishida, Junich; Shimizu, Yuichi; Kawakami, Hiroshi; Suda, Goki; Muranaka, Tetsuhito; Komatsu, Yoshito; Asaka, Masahiro; Sakamoto, Naoya

    2017-03-01

    RNA-binding motif 5 is a putative tumor suppressor gene that modulates cell cycle arrest and apoptosis. We recently demonstrated that RNA-binding motif 5 inhibits cell growth through the p53 pathway. This study evaluated the clinical significance of RNA-binding motif 5 expression in gastric cancer and the effects of altered RNA-binding motif 5 expression on cancer biology in gastric cancer cells. RNA-binding motif 5 protein expression was evaluated by immunohistochemistry using the surgical specimens of 106 patients with gastric cancer. We analyzed the relationships of RNA-binding motif 5 expression with clinicopathological parameters and patient prognosis. We further explored the effects of RNA-binding motif 5 downregulation with short hairpin RNA on cell growth and p53 signaling in MKN45 gastric cancer cells. Immunohistochemistry revealed that RNA-binding motif 5 expression was decreased in 29 of 106 (27.4%) gastric cancer specimens. Decreased RNA-binding motif 5 expression was correlated with histological differentiation, depth of tumor infiltration, nodal metastasis, tumor-node-metastasis stage, and prognosis. RNA-binding motif 5 silencing enhanced gastric cancer cell proliferation and decreased p53 transcriptional activity in reporter gene assays. Conversely, restoration of RNA-binding motif 5 expression suppressed cell growth and recovered p53 transactivation in RNA-binding motif 5-silenced cells. Furthermore, RNA-binding motif 5 silencing reduced the messenger RNA and protein expression of the p53 target gene p21. Our results suggest that RNA-binding motif 5 downregulation is involved in gastric cancer progression and that RNA-binding motif 5 behaves as a tumor suppressor gene in gastric cancer.

  11. Identifying DNA Binding Motifs by Combining Data from Different Sources

    SciTech Connect

    Mao, Linyong; Resat, Haluk; Nagib Callaos; Katsuhisa Horimoto; Jake Chen; Amy Sze Chan

    2004-07-19

    A transcription factor regulates the expression of its target genes by binding to their operator regions. It functions by affecting the interactions between RNA polymerases and the gene's promoter. Many transcription factors bind to their targets by recognizing a specific DNA sequence pattern, which is referred to as a consensus sequence or a motif. Since it would remove the possible biases, combining biological data from different sources can be expected to improve the quality of the information extracted from the biological data. We analyzed the microarray gene expression data and the organism's genome sequence jointly to determine the transcription factor recognition sequences with more accuracy. Utilizing such a data integration approach, we have investigated the regulation of the photosynthesis genes of the purple non-sulphur photosynthetic bacterium Rhodobacter sphaeroides. The photosynthesis genes in this organism are tightly regulated as a function of environmental growth conditions by three major regulatory systems, PrrB/PrrA, AppA/PpsR and FnrL. In this study, we have detected a previously undefined PrrA consensus sequence, improved the previously known DNA-binding motif of PpsR, and confirmed the consensus sequence of the global regulator FnrL.

  12. Bartonella henselae Pap31, an Extracellular Matrix Adhesin, Binds the Fibronectin Repeat III13 Module

    PubMed Central

    Dabo, S. M.; Confer, A. W.; Anderson, B. E.; Gupta, Snehalata

    2006-01-01

    Bartonella henselae wound-associated infections suggest involvement of extracellular matrix molecules in adhesion and invasion. Pap31 was previously identified as a hemin-binding protein. Our recent studies suggest the protein is an adhesin that is recognized by the host's immune systems. In this study we examined the interactions of B. henselae Pap31 with fibronectin (Fn), heparin (Hep), and human umbilical vein endothelial cells (HUVECs). The cloned gene was expressed in Escherichia coli, and the purified Pap31 protein elicited strong antibody responses in mice and was reactive with rabbit anti-live B. henselae and mouse anti-Pap31 antibodies by Western blotting. Pap31 bound to immobilized Fn and to HUVECs in a dose-dependent manner and to Hep. Fn fragment-binding assays identified the Hep-1 and Hep-2 binding domains of human Fn and in particular the 12-13FnIII repeat module as primary binding sites for this adhesin. Furthermore, Pap31 binding to the above Fn fragments could be inhibited by Hep, suggesting a common binding site involving the 13FnIII repeat module on the Hep-2 domain of Fn. Adherence of intact B. henselae to HUVECs was inhibited by increasing concentrations of anti-Pap31 antibodies. In addition, purified Pap31 coprecipitated effectively with Fn and anti-Fn antibodies. Taken together, these data suggest that Pap31 is an Fn-binding protein mediating the B. henselae-host interaction(s), and they implicate the 13FnIII repeat module as an important binding site for this adhesin on the Fn molecule. These interactions may be important initial steps leading to bacterial attachment and colonization that promote the establishment of B. henselae infections in vivo. PMID:16622186

  13. Fibronectin Growth Factor-Binding Domains Are Required for Fibroblast Survival

    PubMed Central

    Lin, Fubao; Ren, Xiang-Dong; Pan, Zhi; Macri, Lauren; Zong, Wei-Xing; Tonnesen, Marcia G.; Rafailovich, Miriam; Bar-Sagi, Dafna; Clark, Richard A.F.

    2011-01-01

    Fibronectin (FN) is required for embryogenesis, morphogenesis, and wound repair, and its Arg–Gly–Asp-containing central cell-binding domain (CCBD) is essential for mesenchymal cell survival and growth. Here, we demonstrate that FN contains three growth factor-binding domains (FN-GFBDs) that bind platelet-derived growth factor-BB (PDGF-BB), a potent fibroblast survival and mitogenic factor. These sites bind PDGF-BB with dissociation constants of 10–100 nm. FN-null cells cultured on recombinant CCBD (FNIII8–11) without a FN-GFBD demonstrated minimal metabolism and underwent autophagy at 24 hours, followed by apoptosis at 72 hours, even in the presence of PDGF-BB. In contrast, FN-null cells plated on FNIII8–11 contiguous with FN-GFBD survived without, and proliferated with, PDGF-BB. FN-null cell survival on FNIII8–11 and noncontiguous arrays of FN-GFBDs required these domains to be adsorbed on the same surface, suggesting the existence of a mesenchymal cell-extracellular matrix synapse. Thus, fibroblast survival required GF stimulation in the presence of a FN-GFBD, as well as adhesion to FN through the CCBD. The findings that fibroblast survival is dependent on FN-GFBD underscore the critical importance of pericellular matrix for cell survival and have significant implications for cutaneous wound healing and regeneration. PMID:20811396

  14. Fibronectin-binding protein of Borrelia hermsii expressed in the blood of mice with relapsing fever.

    PubMed

    Lewis, Eric R G; Marcsisin, Renee A; Campeau Miller, Shelley A; Hue, Fong; Phillips, April; Aucoin, David P; Barbour, Alan G

    2014-06-01

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

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

    PubMed Central

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

    2014-01-01

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

  16. Identifying combinatorial regulation of transcription factors and binding motifs

    PubMed Central

    Kato, Mamoru; Hata, Naoya; Banerjee, Nilanjana; Futcher, Bruce; Zhang, Michael Q

    2004-01-01

    Background Combinatorial interaction of transcription factors (TFs) is important for gene regulation. Although various genomic datasets are relevant to this issue, each dataset provides relatively weak evidence on its own. Developing methods that can integrate different sequence, expression and localization data have become important. Results Here we use a novel method that integrates chromatin immunoprecipitation (ChIP) data with microarray expression data and with combinatorial TF-motif analysis. We systematically identify combinations of transcription factors and of motifs. The various combinations of TFs involved multiple binding mechanisms. We reconstruct a new combinatorial regulatory map of the yeast cell cycle in which cell-cycle regulation can be drawn as a chain of extended TF modules. We find that the pairwise combination of a TF for an early cell-cycle phase and a TF for a later phase is often used to control gene expression at intermediate times. Thus the number of distinct times of gene expression is greater than the number of transcription factors. We also see that some TF modules control branch points (cell-cycle entry and exit), and in the presence of appropriate signals they can allow progress along alternative pathways. Conclusions Combining different data sources can increase statistical power as demonstrated by detecting TF interactions and composite TF-binding motifs. The original picture of a chain of simple cell-cycle regulators can be extended to a chain of composite regulatory modules: different modules may share a common TF component in the same pathway or a TF component cross-talking to other pathways. PMID:15287978

  17. Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment.

    PubMed

    Clark, R A; Wikner, N E; Doherty, D E; Norris, D A

    1988-08-25

    Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding

  18. The helix bundle: A reversible lipid binding motif

    PubMed Central

    Narayanaswami, Vasanthy; Kiss, Robert S.; Weers, Paul M.M.

    2009-01-01

    Apolipoproteins are the protein components of lipoproteins that have the innate ability to inter convert between a lipid-free and a lipid-bound form in a facile manner, a remarkable property conferred by the helix bundle motif. Composed of a series of four or five amphipathic α-helices that fold to form a helix bundle, this motif allows the en face orientation of the hydrophobic faces of the α-helices in the protein interior in the lipid-free state. A conformational switch then permits helix-helix interactions to be substituted by helix-lipid interactions upon lipid binding interaction. This review compares the apolipoprotein high resolution structures and the factors that trigger this switch in insect apolipophorin III and the mammalian apolipoproteins, apolipoprotein E and apolipoprotein A-I, pointing out the commonalities and key differences in the mode of lipid interaction. Further insights into the lipid bound conformation of apolipoproteins are required to fully understand their functional role under physiological conditions. PMID:19770066

  19. The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

    PubMed

    Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

    2014-01-01

    Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile.

  20. Fibronectin peptides that bind PDGF-BB enhance survival of cells and tissue under stress

    PubMed Central

    Lin, Fubao; Zhu, Jia; Tonnesen, Marcia G.; Taira, Breena R.; McClain, Steve A.; Singer, Adam J.; Clark, Richard A.F.

    2013-01-01

    Stressors after injury from a multitude of factors can lead to cell death. We have identified four fibronectin (FN) peptides, two from the first FN type III repeat (FNIII1), one from the 13th FN type III repeat (FNIII13), and one from FN variable region (IIICS), that when tethered to a surface acted as platelet-derived growth factor-BB (PDGF-BB) enhancers to promote cell survival. One of the FNIII1 peptides and its smallest (14mer) bioactive form (P12) were also active in solution. Specifically, P12 bound PDGF-BB (KD = 200nM), enhanced adult human dermal fibroblast (AHDF) survival under serum starvation, oxidative or endoplasmic reticulum (ER) stressors, and limited burn injury progression in a rat hot comb model. Furthermore, P12 inhibited ER stress-induced c-Jun N-terminal kinase (JNK) activation. Although many growth factors have been found to bind FN directly or indirectly, this is the first report to identify peptide sequences of growth factor-binding sites in FN. The finding of these novel peptides further delineated how the extracellular matrix protein FN can support cell survival. Since the peptide P12 is active in either soluble form or tethered to a substrate, it will have multifactorial uses as a bioactive in tissue engineering. PMID:24126844

  1. Manuka honey inhibits the development of Streptococcus pyogenes biofilms and causes reduced expression of two fibronectin binding proteins.

    PubMed

    Maddocks, Sarah E; Lopez, Marta Salinas; Rowlands, Richard S; Cooper, Rose A

    2012-03-01

    Streptococcus pyogenes (group A Streptococcus; GAS) is always of clinical significance in wounds where it can initiate infection, destroy skin grafts and persist as a biofilm. Manuka honey has broad spectrum antimicrobial activity and its use in the clinical setting is beginning to gain acceptance with the continuing emergence of antibiotic resistance and the inadequacy of established systemic therapies; novel inhibitors may affect clinical practice. In this study, the effect of manuka honey on S. pyogenes (M28) was investigated in vitro with planktonic and biofilm cultures using MIC, MBC, microscopy and aggregation efficiency. Bactericidal effects were found in both planktonic cultures and biofilms, although higher concentrations of manuka honey were needed to inhibit biofilms. Abrogation of adherence and intercellular aggregation was observed. Manuka honey permeated 24 h established biofilms of S. pyogenes, resulting in significant cell death and dissociation of cells from the biofilm. Sublethal concentrations of manuka honey effectively prevented the binding of S. pyogenes to the human tissue protein fibronectin, but did not inhibit binding to fibrinogen. The observed inhibition of fibronectin binding was confirmed by a reduction in the expression of genes encoding two major fibronectin-binding streptococcal surface proteins, Sof and SfbI. These findings indicate that manuka honey has potential in the topical treatment of wounds containing S. pyogenes.

  2. Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease.

    PubMed Central

    McGavin, M J; Zahradka, C; Rice, K; Scott, J E

    1997-01-01

    The amount of cell surface fibronectin (Fn)-binding protein (FnBP) adhesin expressed by Staphylococcus aureus is maximal during exponential growth but disappears rapidly as the culture progresses into stationary phase. To identify factors responsible for the loss of cell surface FnBP, a culture of S. aureus L170, which shows high levels of Fn binding, was supplemented at the time of inoculation with concentrated stationary-phase supernatant from S. aureus L530, a strain which binds Fn poorly. The resulting exponential-phase cells were devoid of FnBP. The factor responsible for this activity was purified from the culture supernatant and identified as V8 protease. When cultured with 375 ng of exogenous V8 protease ml(-1), exponential-phase cells of S. aureus L170 were devoid of cell surface FnBP, and concentrations as low as 23 ng x ml(-1) resulted in reduced amounts of FnBP. Addition of the protease inhibitor alpha2-macroglobulin to the culture medium prevented the growth-phase-dependent loss of cell surface FnBP, whereas growth with exogenous V8 protease resulted in reduced adherence to the solid-phase N-terminal fragment of Fn and to the extracellular matrix synthesized by fetal rabbit lung fibroblasts. Although FnBP was extremely sensitive to V8 protease, exogenous protease did not exert a significant influence on the amount of cell surface protein A. However, a limited number of other high-molecular-weight cell surface proteins were also sensitive to V8 protease. Therefore, both the adhesive phenotype and cell surface protein profile of S. aureus can be modified by V8 protease activity. PMID:9199429

  3. Structural and functional analysis of an anchorless fibronectin-binding protein FBPS from Gram-positive bacterium Streptococcus suis

    PubMed Central

    Musyoki, Abednego Moki; Shi, Zhongyu; Xuan, Chunling; Lu, Guangwen; Qi, Jianxun; Gao, Feng; Zheng, Beiwen; Zhang, Qiangmin; Li, Yan; Haywood, Joel; Liu, Cuihua; Yan, Jinghua; Shi, Yi; Gao, George F.

    2016-01-01

    The anchorless fibronectin-binding proteins (FnBPs) are a group of important virulence factors for which the structures are not available and the functions are not well defined. In this study we performed comprehensive studies on a prototypic member of this group: the fibronectin-/fibrinogen-binding protein from Streptococcus suis (FBPS). The structures of the N- and C-terminal halves (FBPS-N and FBPS-C), which together cover the full-length protein in sequence, were solved at a resolution of 2.1 and 2.6 Å, respectively, and each was found to be composed of two domains with unique folds. Furthermore, we have elucidated the organization of these domains by small-angle X-ray scattering. We further showed that the fibronectin-binding site is located in FBPS-C and that FBPS promotes the adherence of S. suis to host cells by attaching the bacteria via FBPS-N. Finally, we demonstrated that FBPS functions both as an adhesin, promoting S. suis attachment to host cells, and as a bacterial factor, activating signaling pathways via β1 integrin receptors to induce chemokine production. PMID:27834729

  4. Magnesium-binding architectures in RNA crystal structures: validation, binding preferences, classification and motif detection

    PubMed Central

    Zheng, Heping; Shabalin, Ivan G.; Handing, Katarzyna B.; Bujnicki, Janusz M.; Minor, Wladek

    2015-01-01

    The ubiquitous presence of magnesium ions in RNA has long been recognized as a key factor governing RNA folding, and is crucial for many diverse functions of RNA molecules. In this work, Mg2+-binding architectures in RNA were systematically studied using a database of RNA crystal structures from the Protein Data Bank (PDB). Due to the abundance of poorly modeled or incorrectly identified Mg2+ ions, the set of all sites was comprehensively validated and filtered to identify a benchmark dataset of 15 334 ‘reliable’ RNA-bound Mg2+ sites. The normalized frequencies by which specific RNA atoms coordinate Mg2+ were derived for both the inner and outer coordination spheres. A hierarchical classification system of Mg2+ sites in RNA structures was designed and applied to the benchmark dataset, yielding a set of 41 types of inner-sphere and 95 types of outer-sphere coordinating patterns. This classification system has also been applied to describe six previously reported Mg2+-binding motifs and detect them in new RNA structures. Investigation of the most populous site types resulted in the identification of seven novel Mg2+-binding motifs, and all RNA structures in the PDB were screened for the presence of these motifs. PMID:25800744

  5. Fibronectin Binding Proteins SpsD and SpsL Both Support Invasion of Canine Epithelial Cells by Staphylococcus pseudintermedius

    PubMed Central

    Pietrocola, Giampiero; Gianotti, Valentina; Richards, Amy; Nobile, Giulia; Geoghegan, Joan A.; Rindi, Simonetta; Monk, Ian R.; Bordt, Andrea S.; Foster, Timothy J.; Fitzgerald, J. Ross

    2015-01-01

    In this study, we investigated the cell wall-anchored fibronectin-binding proteins SpsD and SpsL from the canine commensal and pathogen Staphylococcus pseudintermedius for their role in promoting bacterial invasion of canine progenitor epidermal keratinocytes (CPEK). Invasion was examined by the gentamicin protection assay and fluorescence microscopy. An ΔspsD ΔspsL mutant of strain ED99 had a dramatically reduced capacity to invade CPEK monolayers, while no difference in the invasion level was observed with single mutants. Lactococcus lactis transformed with plasmids expressing SpsD and SpsL promoted invasion, showing that both proteins are important. Soluble fibronectin was required for invasion, and an RGD-containing peptide or antibodies recognizing the integrin α5β1 markedly reduced invasion, suggesting an important role for the integrin in this process. Src kinase inhibitors effectively blocked internalization, suggesting a functional role for the kinase in invasion. In order to identify the minimal fibronectin-binding region of SpsD and SpsL involved in the internalization process, recombinant fragments of both proteins were produced. The SpsD520–846 and SpsL538–823 regions harboring the major fibronectin-binding sites inhibited S. pseudintermedius internalization. Finally, the effects of staphylococcal invasion on the integrity of different cell lines were examined. Because SpsD and SpsL are critical factors for adhesion and invasion, blocking these processes could provide a strategy for future approaches to treating infections. PMID:26238710

  6. Fibronectin Binding Proteins SpsD and SpsL Both Support Invasion of Canine Epithelial Cells by Staphylococcus pseudintermedius.

    PubMed

    Pietrocola, Giampiero; Gianotti, Valentina; Richards, Amy; Nobile, Giulia; Geoghegan, Joan A; Rindi, Simonetta; Monk, Ian R; Bordt, Andrea S; Foster, Timothy J; Fitzgerald, J Ross; Speziale, Pietro

    2015-10-01

    In this study, we investigated the cell wall-anchored fibronectin-binding proteins SpsD and SpsL from the canine commensal and pathogen Staphylococcus pseudintermedius for their role in promoting bacterial invasion of canine progenitor epidermal keratinocytes (CPEK). Invasion was examined by the gentamicin protection assay and fluorescence microscopy. An ΔspsD ΔspsL mutant of strain ED99 had a dramatically reduced capacity to invade CPEK monolayers, while no difference in the invasion level was observed with single mutants. Lactococcus lactis transformed with plasmids expressing SpsD and SpsL promoted invasion, showing that both proteins are important. Soluble fibronectin was required for invasion, and an RGD-containing peptide or antibodies recognizing the integrin α5β1 markedly reduced invasion, suggesting an important role for the integrin in this process. Src kinase inhibitors effectively blocked internalization, suggesting a functional role for the kinase in invasion. In order to identify the minimal fibronectin-binding region of SpsD and SpsL involved in the internalization process, recombinant fragments of both proteins were produced. The SpsD520-846 and SpsL538-823 regions harboring the major fibronectin-binding sites inhibited S. pseudintermedius internalization. Finally, the effects of staphylococcal invasion on the integrity of different cell lines were examined. Because SpsD and SpsL are critical factors for adhesion and invasion, blocking these processes could provide a strategy for future approaches to treating infections.

  7. Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sites

    PubMed Central

    Klotzsch, Enrico; Smith, Michael L.; Kubow, Kristopher E.; Muntwyler, Simon; Little, William C.; Beyeler, Felix; Gourdon, Delphine; Nelson, Bradley J.; Vogel, Viola

    2009-01-01

    Rather than maximizing toughness, as needed for silk and muscle titin fibers to withstand external impact, the much softer extracellular matrix fibers made from fibronectin (Fn) can be stretched by cell generated forces and display extraordinary extensibility. We show that Fn fibers can be extended more than 8-fold (>700% strain) before 50% of the fibers break. The Young's modulus of single fibers, given by the highly nonlinear slope of the stress-strain curve, changes orders of magnitude, up to MPa. Although many other materials plastically deform before they rupture, evidence is provided that the reversible breakage of force-bearing backbone hydrogen bonds enables the large strain. When tension is released, the nano-sized Fn domains first contract in the crowded environment of fibers within seconds into random coil conformations (molten globule states), before the force-bearing hydrogen bond networks that stabilize the domain's secondary structures are reestablished within minutes (double exponential). The exposure of cryptic binding sites on Fn type III modules increases steeply upon stretching. Thus fiber extension steadily up-regulates fiber rigidity and cryptic epitope exposure, both of which are known to differentially alter cell behavior. Finally, since stress-strain relationships cannot directly be measured in native extracellular matrix (ECM), the stress-strain curves were correlated with stretch-induced alterations of intramolecular fluorescence resonance energy transfer (FRET) obtained from trace amounts of Fn probes (mechanical strain sensors) that can be incorporated into native ECM. Physiological implications of the extraordinary extensibility of Fn fibers and contraction kinetics are discussed. PMID:19826086

  8. Fibronectin and atherosclerosis.

    PubMed

    Stenman, S; von Smitten, K; Vaheri, A

    1980-01-01

    Fibronectin is a polymorphic glycoprotein of plasma, other body fluids and connective tissue, and it occurs in an insoluble and a soluble form. Insoluble fibronectin is found associated with basement membranes and in loose connective tissue matrix as well as in the pericellular matrix formed around cultured adherent cells, such as endothelial, fibroblastic and smooth muscle cells. In these positions fibronectin apparently functions as a substrate for cell attachment and as a scaffold for cell migration and movement. Soluble fibronectin, present e.g. in the circulation (300 micronm/ml) exhibits some important interations with other proteins. It is covalently cross-linked to fibrin during thrombus formation and binds to collagen. Fibronectin is released from platelets during their aggregation and soluble fibronectin potentiates the action of plasminogen activator. We have detected fibronectin in the sub-endothelium, in the matrix of smooth muscle cells of the media and in the adventitia of arteries. By using immunohistological techniques we have further found that fibronectin is prominent in atherosclerotic lesions of the intima, especially in developing fibrous plaques. Fibronectin was also prominent in experimentally induced atherosclerotic lesions. These findings suggest that fibronectin is an indicator of connective tissue formation in atherosclerotic processes and that the protein can have a role in their pathogenesis.

  9. Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated

    PubMed Central

    2010-01-01

    Background DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS") but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) not to be biological transcription factor binding sites ("empirical TFBS"). We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. Results Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. Conclusions Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation. PMID:20875111

  10. SarA, a global regulator of virulence determinants in Staphylococcus aureus, binds to a conserved motif essential for sar-dependent gene regulation.

    PubMed

    Chien, Y; Manna, A C; Projan, S J; Cheung, A L

    1999-12-24

    The expression of many virulence determinants in Staphylococcus aureus including alpha-hemolysin-, protein A-, and fibronectin-binding proteins is controlled by global regulatory loci such as sar and agr. In addition to controlling target gene expression via agr (e.g. alpha-hemolysin), the sar locus can also regulate target gene transcription via agr-independent mechanisms. In particular, we have found that SarA, the major regulatory protein encoded within sar, binds to a conserved sequence, homologous to the SarA-binding site on the agr promoter, upstream of the -35 promoter boxes of several target genes including hla (alpha-hemolysin gene), spa (protein A gene), fnb (fibronectin-binding protein genes), and sec (enterotoxin C gene). Deletion of the SarA recognition motif in the promoter regions of agr and hla in shuttle plasmids rendered the transcription of these genes undetectable in agr and hla mutants, respectively. Likewise, the transcription activity of spa (a gene normally repressed by sar), as measured by a XylE reporter fusion assay, became derepressed in a wild type strain containing a shuttle plasmid in which the SarA recognition site had been deleted from the spa promoter region. However, DNase I footprinting assays demonstrated that the SarA-binding region on the spa and hla promoter is more extensive than the predicted consensus sequence, thus raising the possibility that the consensus sequence is an activation site within a larger binding region. Because the sar and agr regulate an assortment of virulence factors in S. aureus, we propose, based on our data, a unifying hypothesis for virulence gene activation in S. aureus whereby SarA is a regulatory protein that binds to its consensus SarA recognition motif to activate (e.g. hla) or repress (e.g. spa) the transcription of sar target genes, thus accounting for both agr-dependent and agr-independent mode of regulation.

  11. Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences

    PubMed Central

    Wright, Elisé P.; Day, Henry A.; Ibrahim, Ali M.; Kumar, Jeethendra; Boswell, Leo J. E.; Huguin, Camille; Stevenson, Clare E. M.; Pors, Klaus; Waller, Zoë A. E.

    2016-01-01

    There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences. PMID:28004744

  12. Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences

    NASA Astrophysics Data System (ADS)

    Wright, Elisé P.; Day, Henry A.; Ibrahim, Ali M.; Kumar, Jeethendra; Boswell, Leo J. E.; Huguin, Camille; Stevenson, Clare E. M.; Pors, Klaus; Waller, Zoë A. E.

    2016-12-01

    There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences.

  13. Analysis of Genomic Sequence Motifs for Deciphering Transcription Factor Binding and Transcriptional Regulation in Eukaryotic Cells

    PubMed Central

    Boeva, Valentina

    2016-01-01

    Eukaryotic genomes contain a variety of structured patterns: repetitive elements, binding sites of DNA and RNA associated proteins, splice sites, and so on. Often, these structured patterns can be formalized as motifs and described using a proper mathematical model such as position weight matrix and IUPAC consensus. Two key tasks are typically carried out for motifs in the context of the analysis of genomic sequences. These are: identification in a set of DNA regions of over-represented motifs from a particular motif database, and de novo discovery of over-represented motifs. Here we describe existing methodology to perform these two tasks for motifs characterizing transcription factor binding. When applied to the output of ChIP-seq and ChIP-exo experiments, or to promoter regions of co-modulated genes, motif analysis techniques allow for the prediction of transcription factor binding events and enable identification of transcriptional regulators and co-regulators. The usefulness of motif analysis is further exemplified in this review by how motif discovery improves peak calling in ChIP-seq and ChIP-exo experiments and, when coupled with information on gene expression, allows insights into physical mechanisms of transcriptional modulation. PMID:26941778

  14. Systematic discovery and characterization of regulatory motifs in ENCODE TF binding experiments

    PubMed Central

    Kheradpour, Pouya; Kellis, Manolis

    2014-01-01

    Recent advances in technology have led to a dramatic increase in the number of available transcription factor ChIP-seq and ChIP-chip data sets. Understanding the motif content of these data sets is an important step in understanding the underlying mechanisms of regulation. Here we provide a systematic motif analysis for 427 human ChIP-seq data sets using motifs curated from the literature and also discovered de novo using five established motif discovery tools. We use a systematic pipeline for calculating motif enrichment in each data set, providing a principled way for choosing between motif variants found in the literature and for flagging potentially problematic data sets. Our analysis confirms the known specificity of 41 of the 56 analyzed factor groups and reveals motifs of potential cofactors. We also use cell type-specific binding to find factors active in specific conditions. The resource we provide is accessible both for browsing a small number of factors and for performing large-scale systematic analyses. We provide motif matrices, instances and enrichments in each of the ENCODE data sets. The motifs discovered here have been used in parallel studies to validate the specificity of antibodies, understand cooperativity between data sets and measure the variation of motif binding across individuals and species. PMID:24335146

  15. Induction of Fibronectin-Binding Proteins and Increased Adhesion of Quinolone-Resistant Staphylococcus aureus by Subinhibitory Levels of Ciprofloxacin

    PubMed Central

    Bisognano, Carmelo; Vaudaux, Pierre; Rohner, Peter; Lew, Daniel P.; Hooper, David C.

    2000-01-01

    We recently reported that strain EN1252a, a fluoroquinolone-resistant derivative of Staphylococcus aureus NCTC8325 with mutations in grlA and gyrA, expressed increased levels of fibronectin-binding proteins (FnBPs) and showed a significantly higher attachment to fibronectin-coated polymer surfaces after growth in the presence of subinhibitory concentrations of ciprofloxacin. The present study evaluated the occurrence and frequency of fluoroquinolone-induced FnBP-mediated adhesion in clinical isolates of fluoroquinolone-resistant methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). Eight of ten MRSA isolates and four of six MSSA isolates with grlA and gyrA mutations exhibited significant increases in attachment to fibronectin-coated surfaces after growth in the presence of one-quarter the MIC of ciprofloxacin. Fluoroquinolone-induced FnBP-mediated adhesion of one clinical MRSA strain and the double mutant strain EN1252a also occurred on coverslips removed from the subcutaneous space of guinea pigs. For strain EN1252a, the regulation of fnb transcription by sub-MICs of ciprofloxacin was studied on reporter plasmids carrying fnb-luxAB fusions. One-quarter of the MIC of ciprofloxacin significantly increased fnbB, but not fnbA, promoter activity of the fluoroquinolone-resistant mutant but not its fluoroquinolone-susceptible parent ISP794. This response was abolished by pretreatment with rifampin, indicating an effect at the level of transcription. Activation of the fnbB promoter was not due to an indirect effect of ciprofloxacin on growth rate and still occurred in an agr mutant of strain EN1252a. These data suggest that sub-MIC levels of ciprofloxacin activate the fnbB promoter of some laboratory and clinical isolates, thus contributing to increased production of FnBP(s) and leading to higher levels of bacterial attachment to fibronectin-coated or subcutaneously implanted coverslips. PMID:10817688

  16. 12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells

    SciTech Connect

    Shiba, Yoshiki; Sasaki, Yasuto; Kanno, Yoshinobu )

    1988-10-01

    The effect of a tumor-promoting phorbol ester on the binding of fibronectin-coated beads to 3T3-L1 cells was studied to clarify the relationship between the binding of fibronectin to the cells, cell adhesion, and the organization of actin filaments. Interference-reflection microscopy revealed focal contacts of 3T3-L1 cells with the substratum. Stress fibers observed after rhodomine-phalloidin staining were well-developed in the cells. Treatment of the cells for 20 min with 12-O-tetradecanoylphorbol-13-acetate (TPA), but not with phorbol, disrupted focal contacts and caused a reorganization of stress fibers to generate actin ribbons. Treatment of the cells with TPA enhanced the binding of beads coated with human plasma fibronectin to the cells, as observed after incubation for 6 h with the beads. The TPA-induced increase in the percentage of cells with bound beads was dependent on the duration of treatment with TPA and on the concentration of TPA. Treatment of the cells with TPA also enhanced proliferation of cells in a dose-dependent manner. Furthermore, treatment of the cells with phorbol did not enhance the binding of beads coated with fibronectin. These results suggest that TPA specifically enhances the binding of fibronectin-coated beads to 3T3-L1 cells, and that TPA-induced binding of the beads may be related to disruption of focal contacts and reorganization of actin filaments.

  17. The Verrucomicrobia LexA-Binding Motif: Insights into the Evolutionary Dynamics of the SOS Response

    PubMed Central

    Erill, Ivan; Campoy, Susana; Kılıç, Sefa; Barbé, Jordi

    2016-01-01

    The SOS response is the primary bacterial mechanism to address DNA damage, coordinating multiple cellular processes that include DNA repair, cell division, and translesion synthesis. In contrast to other regulatory systems, the composition of the SOS genetic network and the binding motif of its transcriptional repressor, LexA, have been shown to vary greatly across bacterial clades, making it an ideal system to study the co-evolution of transcription factors and their regulons. Leveraging comparative genomics approaches and prior knowledge on the core SOS regulon, here we define the binding motif of the Verrucomicrobia, a recently described phylum of emerging interest due to its association with eukaryotic hosts. Site directed mutagenesis of the Verrucomicrobium spinosum recA promoter confirms that LexA binds a 14 bp palindromic motif with consensus sequence TGTTC-N4-GAACA. Computational analyses suggest that recognition of this novel motif is determined primarily by changes in base-contacting residues of the third alpha helix of the LexA helix-turn-helix DNA binding motif. In conjunction with comparative genomics analysis of the LexA regulon in the Verrucomicrobia phylum, electrophoretic shift assays reveal that LexA binds to operators in the promoter region of DNA repair genes and a mutagenesis cassette in this organism, and identify previously unreported components of the SOS response. The identification of tandem LexA-binding sites generating instances of other LexA-binding motifs in the lexA gene promoter of Verrucomicrobia species leads us to postulate a novel mechanism for LexA-binding motif evolution. This model, based on gene duplication, successfully addresses outstanding questions in the intricate co-evolution of the LexA protein, its binding motif and the regulatory network it controls. PMID:27489856

  18. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.

    PubMed Central

    Yang, B; Yang, B L; Savani, R C; Turley, E A

    1994-01-01

    We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R431, spaced seven amino acids apart, are critical for HA binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA--Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X7)B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, R431 and K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7)B motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses

  19. Structural basis for the binding of tryptophan-based motifs by δ-COP

    PubMed Central

    Suckling, Richard J.; Poon, Pak Phi; Travis, Sophie M.; Majoul, Irina V.; Hughson, Frederick M.; Evans, Philip R.; Duden, Rainer; Owen, David J.

    2015-01-01

    Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. PMID:26578768

  20. Structural basis for the binding of tryptophan-based motifs by δ-COP.

    PubMed

    Suckling, Richard J; Poon, Pak Phi; Travis, Sophie M; Majoul, Irina V; Hughson, Frederick M; Evans, Philip R; Duden, Rainer; Owen, David J

    2015-11-17

    Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ'ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.

  1. WordSpy: identifying transcription factor binding motifs by building a dictionary and learning a grammar

    PubMed Central

    Wang, Guandong; Yu, Taotao; Zhang, Weixiong

    2005-01-01

    Transcription factor (TF) binding sites or motifs (TFBMs) are functional cis-regulatory DNA sequences that play an essential role in gene transcriptional regulation. Although many experimental and computational methods have been developed, finding TFBMs remains a challenging problem. We propose and develop a novel dictionary based motif finding algorithm, which we call WordSpy. One significant feature of WordSpy is the combination of a word counting method and a statistical model which consists of a dictionary of motifs and a grammar specifying their usage. The algorithm is suitable for genome-wide motif finding; it is capable of discovering hundreds of motifs from a large set of promoters in a single run. We further enhance WordSpy by applying gene expression information to separate true TFBMs from spurious ones, and by incorporating negative sequences to identify discriminative motifs. In addition, we also use randomly selected promoters from the genome to evaluate the significance of the discovered motifs. The output from WordSpy consists of an ordered list of putative motifs and a set of regulatory sequences with motif binding sites highlighted. The web server of WordSpy is available at . PMID:15980501

  2. Small yet effective: the ethylene responsive element binding factor-associated amphiphilic repression (EAR) motif.

    PubMed

    Kagale, Sateesh; Rozwadowski, Kevin

    2010-06-01

    The Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif is a small yet distinct regulatory motif that is conserved in many plant transcriptional regulator (TR) proteins associated with diverse biological functions. We have previously established a list of high-confidence Arabidopsis EAR repressors, the EAR repressome, comprising 219 TRs belonging to 21 different TR families. This class of proteins and the sequence context of the EAR motif exhibited a high degree of conservation across evolutionarily diverse plant species. Our comprehensive genome-wide analysis enabled refining EAR motifs as comprising either LxLxL or DLNxxP. Comparing the representation of these sequence signatures in TRs to that of other repressor motifs we show that the EAR motif is the one most frequently represented, detected in 10 to 25% of the TRs from diverse plant species. The mechanisms involved in regulation of EAR motif function and the cellular fates of EAR repressors are currently not well understood. Our earlier analysis had implicated amino acid residues flanking the EAR motifs in regulation of their functionality. Here, we present additional evidence supporting possible regulation of EAR motif function by phosphorylation of integral or adjacent Ser and/or Thr residues. Additionally, we discuss potential novel roles of EAR motifs in plant-pathogen interaction and processes other than transcriptional repression.

  3. Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

    PubMed

    Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

    2016-11-28

    Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes.

  4. A compendium of RNA-binding motifs for decoding gene regulation

    PubMed Central

    Ray, Debashish; Kazan, Hilal; Cook, Kate B.; Weirauch, Matthew T.; Najafabadi, Hamed S.; Li, Xiao; Gueroussov, Serge; Albu, Mihai; Zheng, Hong; Yang, Ally; Na, Hong; Irimia, Manuel; Matzat, Leah H.; Dale, Ryan K.; Smith, Sarah A.; Yarosh, Christopher A.; Kelly, Seth M.; Nabet, Behnam; Mecenas, Desirea; Li, Weimin; Laishram, Rakesh S.; Qiao, Mei; Lipshitz, Howard D.; Piano, Fabio; Corbett, Anita H.; Carstens, Russ P.; Frey, Brendan J.; Anderson, Richard A.; Lynch, Kristen W.; Penalva, Luiz O. F.; Lei, Elissa P.; Fraser, Andrew G.; Blencowe, Benjamin J.; Morris, Quaid D.; Hughes, Timothy R.

    2014-01-01

    RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes. PMID:23846655

  5. RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities

    PubMed Central

    1990-01-01

    Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with

  6. Peptide-based identification of functional motifs and their binding partners.

    PubMed

    Shelton, Martin N; Huang, Ming Bo; Ali, Syed; Johnson, Kateena; Roth, William; Powell, Michael; Bond, Vincent

    2013-06-30

    Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

  7. LibME-automatic extraction of 3D ligand-binding motifs for mechanistic analysis of protein-ligand recognition.

    PubMed

    He, Wei; Liang, Zhi; Teng, MaiKun; Niu, LiWen

    2016-12-01

    Identifying conserved binding motifs is an efficient way to study protein-ligand recognition. Most 3D binding motifs only contain information from the protein side, and so motifs that combine information from both protein and ligand sides are desired. Here, we propose an algorithm called LibME (Ligand-binding Motif Extractor), which automatically extracts 3D binding motifs composed of the target ligand and surrounding conserved residues. We show that the motifs extracted by LibME for ATP and its analogs are highly similar to well-known motifs reported by previous studies. The superiority of our method to handle flexible ligands was also demonstrated using isocitric acid as an example. Finally, we show that these motifs, together with their visual exhibition, permit better investigating and understanding of protein-ligand recognition process.

  8. Soluble CD163 masks fibronectin-binding protein A-mediated inflammatory activation of Staphylococcus aureus infected monocytes.

    PubMed

    Kneidl, Jessica; Mysore, Vijayashree; Geraci, Jennifer; Tuchscherr, Lorena; Löffler, Bettina; Holzinger, Dirk; Roth, Johannes; Barczyk-Kahlert, Katarzyna

    2014-03-01

    Binding to fibronectin (FN) is a crucial pathogenic factor of Staphylococcus aureus mediated by fibronectin-binding protein A (FnBP-A) and extracellular adherence protein (Eap). Recently, we have shown that binding of soluble CD163 (sCD163) to FN linked to these molecules exhibits anti-microbial effects by enhancing phagocytosis and killing activity of S. aureus-infected monocytes. However, it remained unclear whether sCD163 also influences the monocytic activation status. Using genetically modified staphylococcal strains we now identified FnBP-A, but not Eap, as activator of the inflammatory response of monocytes to infection. FnBP-A-mediated inflammatory activation was masked by sCD163 binding to S. aureus promoting efficient pathogen elimination. Thus, sCD163 protects monocytes from overwhelming activation upon staphylococcal infection by dampening the secretion of pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL-8 and DAMP molecule MRP8/14. Moreover, sCD163 limited expression of pro-apoptotic transcription factor NR4A1 induced during S. aureus infection and inhibited induction of chemokine CXCL2promoting survival of staphylococci in vivo. sCD163-mediated effects were not due to general immunosuppression since MAP kinase activation and ROS production were unaltered during infection of monocytes with sCD163-bound bacteria. Thus, sCD163 promotes a specific defence of the immune system against FnBP-A-mediated inflammatory activation enabling successful pathogen elimination, tissue recovery and resolution of inflammation.

  9. Zinc finger binding motifs do not explain recombination rate variation within or between species of Drosophila.

    PubMed

    Heil, Caiti S S; Noor, Mohamed A F

    2012-01-01

    In humans and mice, the Cys(2)His(2) zinc finger protein PRDM9 binds to a DNA sequence motif enriched in hotspots of recombination, possibly modifying nucleosomes, and recruiting recombination machinery to initiate Double Strand Breaks (DSBs). However, since its discovery, some researchers have suggested that the recombinational effect of PRDM9 is lineage or species specific. To test for a conserved role of PRDM9-like proteins across taxa, we use the Drosophila pseudoobscura species group in an attempt to identify recombination associated zinc finger proteins and motifs. We leveraged the conserved amino acid motifs in Cys(2)His(2) zinc fingers to predict nucleotide binding motifs for all Cys(2)His(2) zinc finger proteins in Drosophila pseudoobscura and identified associations with empirical measures of recombination rate. Additionally, we utilized recombination maps from D. pseudoobscura and D. miranda to explore whether changes in the binding motifs between species can account for changes in the recombination landscape, analogous to the effect observed in PRDM9 among human populations. We identified a handful of potential recombination-associated sequence motifs, but the associations are generally tenuous and their biological relevance remains uncertain. Furthermore, we found no evidence that changes in zinc finger DNA binding explains variation in recombination rate between species. We therefore conclude that there is no protein with a DNA sequence specific human-PRDM9-like function in Drosophila. We suggest these findings could be explained by the existence of a different recombination initiation system in Drosophila.

  10. Mutated Leguminous Lectin Containing a Heparin-Binding like Motif in a Carbohydrate-Binding Loop Specifically Binds to Heparin

    PubMed Central

    Abo, Hirohito; Soga, Keisuke; Tanaka, Atsuhiro; Tateno, Hiroaki; Hirabayashi, Jun; Yamamoto, Kazuo

    2015-01-01

    We previously introduced random mutations in the sugar-binding loops of a leguminous lectin and screened the resulting mutated lectins for novel specificities using cell surface display. Screening of a mutated peanut agglutinin (PNA), revealed a mutated PNA with a distinct preference for heparin. Glycan microarray analyses using the mutated lectin fused to the Fc region of human immunoglobulin, revealed that a particular sulfated glycosaminoglycan (GAG), heparin, had the highest binding affinity for mutated PNA among 97 glycans tested, although wild-type PNA showed affinity towards Galβ1-3GalNAc and similar galactosylated glycans. Further analyses of binding specificity using an enzyme-linked immunoadsorbent assay demonstrated that the mutated PNA specifically binds to heparin, and weakly to de-2-O-sulfated heparin, but not to other GAG chains including de-6-O-sulfated and de-N-sulfated heparins. The mutated PNA had six amino acid substitutions within the eight amino acid-long sugar-binding loop. In this loop, the heparin-binding like motif comprised three arginine residues at positions 124, 128, and 129, and a histidine at position 125 was present. Substitution of each arginine or histidine residue to alanine reduced heparin-binding ability, indicating that all of these basic amino acid residues contributed to heparin binding. Inhibition assay demonstrated that heparin and dextran sulfate strongly inhibited mutated PNA binding to heparin in dose-dependent manner. The mutated PNA could distinguish between CHO cells and proteoglycan-deficient mutant cells. This is the first report establishing a novel leguminous lectin that preferentially binds to highly sulfated heparin and may provide novel GAG-binding probes to distinguish between heterogeneous GAG repeating units. PMID:26714191

  11. Fibronectin regulates calvarial osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Moursi, A. M.; Damsky, C. H.; Lull, J.; Zimmerman, D.; Doty, S. B.; Aota, S.; Globus, R. K.

    1996-01-01

    The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not

  12. The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase

    PubMed Central

    Ding, Hao; Guo, Manhong; Vidhyasagar, Venkatasubramanian; Talwar, Tanu; Wu, Yuliang

    2015-01-01

    Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of nine amino acids (GFXXPXPIQ) with an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase. PMID:26474416

  13. Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure.

    PubMed Central

    Thayer, M M; Ahern, H; Xing, D; Cunningham, R P; Tainer, J A

    1995-01-01

    The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins. Images PMID:7664751

  14. Asbestos fibre length-dependent detachment injury to alveolar epithelial cells in vitro: role of a fibronectin-binding receptor.

    PubMed Central

    Donaldson, K.; Miller, B. G.; Sara, E.; Slight, J.; Brown, R. C.

    1993-01-01

    A short and a long fibre sample of amosite asbestos were tested for their effects on cells of the human Type 2 alveolar epithelial cell-line A549 in vitro. The long amosite sample was found to cause a rapid detachment of the epithelial cells live from their substratum. At the highest dose, on average 28% of the cells present were detached in this way. Studies on the mechanism of the detachment injury showed that it did not involve oxidants since it was not ameliorated by scavengers of active oxygen species. Neither was the effect reduced by treatment of the fibres with the iron chelator Desferal. Treatments reported to increase the interaction between fibres and cells, serum and poly-L-lysine, did not influence the detachment injury, nor did lung lining fluid. Conversely, the fibronectin tripeptide RGD alone could cause detachment which suggested that a fibronectin-binding integrin was involved. This receptor could be reduced in activity by long fibre exposure, leading to detachment. The detaching effect of fibre could be mimicked by the protein kinase C activator PMA, and so the second messenger system of the cell could also be involved. This type of injury could be important in the pathology associated with exposure to long fibres. PMID:8392859

  15. Identification of a binding motif specific to HNF4 by comparative analysis of multiple nuclear receptors

    PubMed Central

    Fang, Bin; Mane-Padros, Daniel; Bolotin, Eugene; Jiang, Tao; Sladek, Frances M.

    2012-01-01

    Nuclear receptors (NRs) regulate gene expression by binding specific DNA sequences consisting of AG[G/T]TCA or AGAACA half site motifs in a variety of configurations. However, those motifs/configurations alone do not adequately explain the diversity of NR function in vivo. Here, a systematic examination of DNA binding specificity by protein-binding microarrays (PBMs) of three closely related human NRs—HNF4α, retinoid X receptor alpha (RXRα) and COUPTF2—reveals an HNF4-specific binding motif (H4-SBM), xxxxCAAAGTCCA, as well as a previously unrecognized polarity in the classical DR1 motif (AGGTCAxAGGTCA) for HNF4α, RXRα and COUPTF2 homodimers. ChIP-seq data indicate that the H4-SBM is uniquely bound by HNF4α but not 10 other NRs in vivo, while NRs PXR, FXRα, Rev-Erbα appear to bind adjacent to H4-SBMs. HNF4-specific DNA recognition and transactivation are mediated by residues Asp69 and Arg76 in the DNA-binding domain; this combination of amino acids is unique to HNF4 among all human NRs. Expression profiling and ChIP data predict ∼100 new human HNF4α target genes with an H4-SBM site, including several Co-enzyme A-related genes and genes with links to disease. These results provide important new insights into NR DNA binding. PMID:22383578

  16. Bayesian multiple-instance motif discovery with BAMBI: inference of recombinase and transcription factor binding sites

    PubMed Central

    Jajamovich, Guido H.; Wang, Xiaodong; Arkin, Adam P.; Samoilov, Michael S.

    2011-01-01

    Finding conserved motifs in genomic sequences represents one of essential bioinformatic problems. However, achieving high discovery performance without imposing substantial auxiliary constraints on possible motif features remains a key algorithmic challenge. This work describes BAMBI—a sequential Monte Carlo motif-identification algorithm, which is based on a position weight matrix model that does not require additional constraints and is able to estimate such motif properties as length, logo, number of instances and their locations solely on the basis of primary nucleotide sequence data. Furthermore, should biologically meaningful information about motif attributes be available, BAMBI takes advantage of this knowledge to further refine the discovery results. In practical applications, we show that the proposed approach can be used to find sites of such diverse DNA-binding molecules as the cAMP receptor protein (CRP) and Din-family site-specific serine recombinases. Results obtained by BAMBI in these and other settings demonstrate better statistical performance than any of the four widely-used profile-based motif discovery methods: MEME, BioProspector with BioOptimizer, SeSiMCMC and Motif Sampler as measured by the nucleotide-level correlation coefficient. Additionally, in the case of Din-family recombinase target site discovery, the BAMBI-inferred motif is found to be the only one functionally accurate from the underlying biochemical mechanism standpoint. C++ and Matlab code is available at http://www.ee.columbia.edu/~guido/BAMBI or http://genomics.lbl.gov/BAMBI/. PMID:21948794

  17. Motif discovery with data mining in 3D protein structure databases: discovery, validation and prediction of the U-shape zinc binding ("Huf-Zinc") motif.

    PubMed

    Maurer-Stroh, Sebastian; Gao, He; Han, Hao; Baeten, Lies; Schymkowitz, Joost; Rousseau, Frederic; Zhang, Louxin; Eisenhaber, Frank

    2013-02-01

    Data mining in protein databases, derivatives from more fundamental protein 3D structure and sequence databases, has considerable unearthed potential for the discovery of sequence motif--structural motif--function relationships as the finding of the U-shape (Huf-Zinc) motif, originally a small student's project, exemplifies. The metal ion zinc is critically involved in universal biological processes, ranging from protein-DNA complexes and transcription regulation to enzymatic catalysis and metabolic pathways. Proteins have evolved a series of motifs to specifically recognize and bind zinc ions. Many of these, so called zinc fingers, are structurally independent globular domains with discontinuous binding motifs made up of residues mostly far apart in sequence. Through a systematic approach starting from the BRIX structure fragment database, we discovered that there exists another predictable subset of zinc-binding motifs that not only have a conserved continuous sequence pattern but also share a characteristic local conformation, despite being included in totally different overall folds. While this does not allow general prediction of all Zn binding motifs, a HMM-based web server, Huf-Zinc, is available for prediction of these novel, as well as conventional, zinc finger motifs in protein sequences. The Huf-Zinc webserver can be freely accessed through this URL (http://mendel.bii.a-star.edu.sg/METHODS/hufzinc/).

  18. A designed DNA binding motif that recognizes extended sites and spans two adjacent major grooves†

    PubMed Central

    Rodríguez, Jéssica; Mosquera, Jesús; García-Fandiño, Rebeca; Vázquez, M. Eugenio; Mascareñas, José L.

    2016-01-01

    We report the rational design of a DNA-binding peptide construct composed of the DNA-contacting regions of two transcription factors (GCN4 and GAGA) linked through an AT-hook DNA anchor. The resulting chimera, which represents a new, non-natural DNA binding motif, binds with high affinity and selectivity to a long composite sequence of 13 base pairs (TCAT-AATT-GAGAG). PMID:27252825

  19. Identification and characterization of the actin-binding motif of phostensin.

    PubMed

    Wang, Tzu-Fan; Lai, Ning-Sheng; Huang, Kuang-Yung; Huang, Hsien-Lu; Lu, Ming-Chi; Lin, Yu-Shan; Chen, Chun-Yu; Liu, Su-Qin; Lin, Ta-Hsien; Huang, Hsien-Bin

    2012-11-28

    Phostensin, a protein phosphatase 1 F-actin cytoskeleton-targeting subunit encoded by KIAA1949, consists of 165 amino acids and caps the pointed ends of actin filaments. Sequence alignment analyses suggest that the C-terminal region of phostensin, spanning residues 129 to 155, contains a consensus actin-binding motif. Here, we have verified the existence of an actin-binding motif in the C-terminal domain of phostensin using colocalization, F-actin co-sedimentation and single filament binding assays. Our data indicate that the N-terminal region of phostensin (1-129) cannot bind to actin filaments and cannot retard the pointed end elongation of gelsolin-actin seeds. Furthermore, the C-terminal region of phostensin (125-165) multiply bind to the sides of actin filaments and lacks the ability to block the pointed end elongation, suggesting that the actin-binding motif is located in the C-terminal region of the phostensin. Further analyses indicate that phostensin binding to the pointed end of actin filament requires N-terminal residues 35 to 51. These results suggest that phostensin might fold into a rigid structure, allowing the N-terminus to sterically hinder the binding of C-terminus to the sides of actin filament, thus rendering phostensin binding to the pointed ends of actin filaments.

  20. Identification of peptide motif that binds to the surface of zirconia.

    PubMed

    Hashimoto, Kazuhiko; Yoshinari, Masao; Matsuzaka, Kenichi; Shiba, Kiyotaka; Inoue, Takashi

    2011-01-01

    A zirconia-binding peptide motif was identified using a peptide phage display system. Yttria stabilized zirconia beads and discs were used as the target. Quartz crystal microbalance was used to monitor the binding of phages to zirconia. Starting from a library of phages displaying random sequences of 12-mer peptides, we repeated cycles of biopanning against zirconia beads. After four cycles of biopanning, we isolated a phage clone Φ#17. DNA sequencing of the corresponding portion of Φ#17 unexpectedly revealed that it displayed a 58-mer peptide (amino acid sequence: WMPSDVDINDPQGGGSRPNLHQPKPAAEAASKKKSENRKVPFYSHSWY-SSMSEDKRGW). We found that Φ#17 had a 300-fold, significantly higher binding affinity for zirconia discs than phages displaying no peptide. In quartz crystal microbalance assay, a rapid increase in energy dissipation was observed from Φ#17 but not from the control phages, indicating that Φ#17 binds to the surface of zirconia via its displayed peptide. We successfully identified a peptide motif that binds zirconia.

  1. Identification of novel proteins secreted by Lactobacillus plantarum that bind to mucin and fibronectin.

    PubMed

    Sánchez, Borja; Schmitter, Jean-Marie; Urdaci, María C

    2009-01-01

    Lactobacillus plantarum is a lactic acid bacterium that can be isolated from a high variety of fermented foods, including dairy products. In the present work, eight novel proteins secreted by three L. plantarum strains have been identified by tandem mass spectrometry. Seven of them were predicted as extracellular proteins containing putative signal peptides. The sixth, identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), is a cytoplasmic protein that has been detected on the surface of several microorganisms. Muramidase and GAPDH were secreted only by the L. plantarum BMCM12 strain. Two other bands present in this strain were not identified, in spite of their yielding good tryptic profiles, suggesting an absence of homolog sequences in the molecular databases. Four of these proteins, including GAPDH, bound to mucin and fibronectin. These proteins might play important roles in the physiology and ecology of this bacterium, notably in the interaction with the human host.

  2. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

    PubMed

    Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

    2016-01-01

    Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  3. The Streptococcus gordonii Adhesin CshA Protein Binds Host Fibronectin via a Catch-Clamp Mechanism*♦

    PubMed Central

    Back, Catherine R.; Sztukowska, Maryta N.; Till, Marisa; Lamont, Richard J.; Jenkinson, Howard F.; Nobbs, Angela H.; Race, Paul R.

    2017-01-01

    Adherence of bacteria to biotic or abiotic surfaces is a prerequisite for host colonization and represents an important step in microbial pathogenicity. This attachment is facilitated by bacterial adhesins at the cell surface. Because of their size and often elaborate multidomain architectures, these polypeptides represent challenging targets for detailed structural and functional characterization. The multifunctional fibrillar adhesin CshA, which mediates binding to both host molecules and other microorganisms, is an important determinant of colonization by Streptococcus gordonii, an oral commensal and opportunistic pathogen of animals and humans. CshA binds the high-molecular-weight glycoprotein fibronectin (Fn) via an N-terminal non-repetitive region, and this protein-protein interaction has been proposed to promote S. gordonii colonization at multiple sites within the host. However, the molecular details of how these two proteins interact have yet to be established. Here we present a structural description of the Fn binding N-terminal region of CshA, derived from a combination of X-ray crystallography, small angle X-ray scattering, and complementary biophysical methods. In vitro binding studies support a previously unreported two-state “catch-clamp” mechanism of Fn binding by CshA, in which the disordered N-terminal domain of CshA acts to “catch” Fn, via formation of a rapidly assembled but also readily dissociable pre-complex, enabling its neighboring ligand binding domain to tightly clamp the two polypeptides together. This study presents a new paradigm for target binding by a bacterial adhesin, the identification of which will inform future efforts toward the development of anti-adhesive agents that target S. gordonii and related streptococci. PMID:27920201

  4. Fibronectin at select sites binds multiple growth factors (GF) and enhances their activity: expansion of the collaborative ECM-GF paradigm

    PubMed Central

    Zhu, Jia; Clark, Richard A.F.

    2013-01-01

    Intensive research has demonstrated that extracellular matrix (ECM) molecules and growth factors (GF) collaborate at many different levels. The ability of ECM to modulate GF signals has important implications in tissue formation and homeostasis as well as novel therapies for acute and chronic wounds. Recently, a number of GF binding sites were identified in fibronectin and were shown to provide another layer of regulation on GF signaling. Here, we review these new findings on fibronectin interaction with GF in context of general ways ECM molecules regulate GF signaling. PMID:24335899

  5. The extended AT-hook is a novel RNA binding motif.

    PubMed

    Filarsky, Michael; Zillner, Karina; Araya, Ingrid; Villar-Garea, Ana; Merkl, Rainer; Längst, Gernot; Németh, Attila

    2015-01-01

    The AT-hook has been defined as a DNA binding peptide motif that contains a glycine-arginine-proline (G-R-P) tripeptide core flanked by basic amino acids. Recent reports documented variations in the sequence of AT-hooks and revealed RNA binding activity of some canonical AT-hooks, suggesting a higher structural and functional variability of this protein domain than previously anticipated. Here we describe the discovery and characterization of the extended AT-hook peptide motif (eAT-hook), in which basic amino acids appear symmetrical mainly at a distance of 12-15 amino acids from the G-R-P core. We identified 80 human and 60 mouse eAT-hook proteins and biochemically characterized the eAT-hooks of Tip5/BAZ2A, PTOV1 and GPBP1. Microscale thermophoresis and electrophoretic mobility shift assays reveal the nucleic acid binding features of this peptide motif, and show that eAT-hooks bind RNA with one order of magnitude higher affinity than DNA. In addition, cellular localization studies suggest a role for the N-terminal eAT-hook of PTOV1 in nucleocytoplasmic shuttling. In summary, our findings classify the eAT-hook as a novel nucleic acid binding motif, which potentially mediates various RNA-dependent cellular processes.

  6. Photonic crystal borax competitive binding carbohydrate sensing motif.

    PubMed

    Cui, Qingzhou; Ward Muscatello, Michelle M; Asher, Sanford A

    2009-05-01

    We developed a photonic crystal sensing method for diol containing species such as carbohydrates based on a poly(vinyl alcohol) (PVA) hydrogel containing an embedded crystalline colloidal array (CCA). The polymerized CCA (PCCA) diffracts visible light. We show that in the presence of borax the diffraction wavelength shifts as the concentration of glucose changes. The diffraction shifts result from the competitive binding of glucose to borate, which reduces the concentration of borate bound to the PVA diols.

  7. Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs

    PubMed Central

    Düvel, Juliane; Bense, Sarina; Möller, Stefan; Bertinetti, Daniela; Schwede, Frank; Morr, Michael; Eckweiler, Denitsa; Genieser, Hans-Gottfried; Jänsch, Lothar; Herberg, Friedrich W.; Frank, Ronald

    2015-01-01

    ABSTRACT High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein. IMPORTANCE In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c

  8. Investigation of structural mimetics of natural phosphate ion binding motifs.

    PubMed

    Kataev, Evgeny A; Shumilova, Tatiana A

    2015-02-16

    Phosphates are ubiquitous in biology and nearly half of all proteins interact with their partners by means of recognition of phosphate residues. Therefore, a better understanding of the phosphate ion binding by peptidic structures is highly desirable. Two new receptors have been designed and synthesized and their anion binding properties in an acetonitrile solution have been determined. The structure of hosts mimics a part of the kinase active site that is responsible for the recognition of the phosphate residue. New hosts contain additional free amino groups with the aim to facilitate coordination of protonated anions, such as dihydrogen phosphate. According to spectrophotometric measurements, stepwise 1:1 and 1:2 binding modes have been observed for both receptors in the presence of acetate, hydrogen sulfate and dihydrogen phosphate. Compared with the acyclic receptor, the macrocyclic receptor has demonstrated a remarkably enhanced selectivity for dihydrogen phosphate over other anions. Fluorometric measurements have revealed different responses of the acyclic and macrocyclic receptors towards anions. However, in both cases, a 5-8 nm hypsochromic shift of fluorescence maximum has been observed upon interaction of acetate and dihydrogen phosphate with receptors.

  9. Inhibition of CD4+ T lymphocyte binding to fibronectin and immune-cell accumulation in inflammatory sites by non-peptidic mimetics of Arg-Gly-Asp.

    PubMed Central

    Hershkoviz, R; Greenspoon, N; Mekori, Y A; Hadari, R; Alon, R; Kapustina, G; Lider, O

    1994-01-01

    The Arg-Gly-Asp (RGD) cell adhesion motif has been demonstrated in various studies to play a pivotal role in leucocyte and platelet interactions with plasma and extracellular matrix (ECM) glycoproteins. The recognition of the RGD sequence is mediated by heterodimeric receptors designated integrins of the beta 1 subfamily, expressed on distinct cell types, including T lymphocytes. We have recently shown that flexible non-peptidic mimetics of RGD, in which the two ionic side groups were separated by a linear spacer of 11 atoms, bound specifically to the platelet integrin alpha 11b beta 3, and inhibited T cell-mediated immune responses. The present study was designed to (i) further characterize the structural requirements for RGD interactions with CD4+ T cells, and (ii) examine the mechanisms by which the RGD mimetics interfere with immune cell reactivity in vivo. We now report that freezing the conformational degrees of freedom in the spacer chain, which fixes the relative orientation of the guanidinium and carboxylate side groups in a favourable manner, results in a higher level of inhibition of T cell binding to immobilized fibronectin, an RGD-containing ECM glycoprotein. In vivo, treatment of mice with relatively low doses of the RGD mimetics, but not the RGD peptide, inhibited the elicitation of an adoptively transferred DTH reaction. This inhibition was achieved by direct impairment of the ability of antigen-primed lymph node cells to migrate and accumulate in inflammatory sites. Hence, we suggest that the design and production of non-peptidic mimetics of RGD offers a novel approach to study defined parameters related to the structure-function requirements of small adhesion epitopes. Furthermore, this approach could be used therapeutically to inhibit pathological processes which depend on RGD recognition. PMID:7905794

  10. Investigating the mechanism of the assembly of FGF1-binding heparan sulfate motifs

    PubMed Central

    Nguyen, Thao Kim Nu; Raman, Karthik; Trana, Vy My; Kuberan, Balagurunathan

    2011-01-01

    Heparan sulfate (HS) chains play crucial biological roles by binding to various signaling molecules including fibroblast growth factors (FGFs). Distinct sulfation patterns of HS chains are required for their binding to FGFs/FGF receptors (FGFRs). These sulfation patterns are putatively regulated by biosynthetic enzyme complexes, called GAGOSOMES, in the Golgi. While the structural requirements of HS-FGF interactions have been described previously, it is still unclear how the FGF-binding motif is assembled in vivo. In this study, we generated HS structures using biosynthetic enzymes in a sequential or concurrent manner to elucidate the potential mechanism by which the FGF1-binding HS motif is assembled. Our results indicate that the HS chains form ternary complexes with FGF1/FGFR when enzymes carry out modifications in a specific manner. PMID:21803043

  11. Selection of peptide entry motifs by bacterial surface display.

    PubMed Central

    Taschner, Sabine; Meinke, Andreas; von Gabain, Alexander; Boyd, Aoife P

    2002-01-01

    Surface display technologies have been established previously to select peptides and polypeptides that interact with purified immobilized ligands. In the present study, we designed and implemented a surface display-based technique to identify novel peptide motifs that mediate entry into eukaryotic cells. An Escherichia coli library expressing surface-displayed peptides was combined with eukaryotic cells and the gentamicin protection assay was performed to select recombinant E. coli, which were internalized into eukaryotic cells by virtue of the displayed peptides. To establish the proof of principle of this approach, the fibronectin-binding motifs of the fibronectin-binding protein A of Staphylococcus aureus were inserted into the E. coli FhuA protein. Surface expression of the fusion proteins was demonstrated by functional assays and by FACS analysis. The fibronectin-binding motifs were shown to mediate entry of the bacteria into non-phagocytic eukaryotic cells and brought about the preferential selection of these bacteria over E. coli expressing parental FhuA, with an enrichment of 100000-fold. Four entry sequences were selected and identified using an S. aureus library of peptides displayed in the FhuA protein on the surface of E. coli. These sequences included novel entry motifs as well as integrin-binding Arg-Gly-Asp (RGD) motifs and promoted a high degree of bacterial entry. Bacterial surface display is thus a powerful tool to effectively select and identify entry peptide motifs. PMID:12144529

  12. ConBind: motif-aware cross-species alignment for the identification of functional transcription factor binding sites

    PubMed Central

    Lelieveld, Stefan H.; Schütte, Judith; Dijkstra, Maurits J.J.; Bawono, Punto; Kinston, Sarah J.; Göttgens, Berthold; Heringa, Jaap; Bonzanni, Nicola

    2016-01-01

    Eukaryotic gene expression is regulated by transcription factors (TFs) binding to promoter as well as distal enhancers. TFs recognize short, but specific binding sites (TFBSs) that are located within the promoter and enhancer regions. Functionally relevant TFBSs are often highly conserved during evolution leaving a strong phylogenetic signal. While multiple sequence alignment (MSA) is a potent tool to detect the phylogenetic signal, the current MSA implementations are optimized to align the maximum number of identical nucleotides. This approach might result in the omission of conserved motifs that contain interchangeable nucleotides such as the ETS motif (IUPAC code: GGAW). Here, we introduce ConBind, a novel method to enhance alignment of short motifs, even if their mutual sequence similarity is only partial. ConBind improves the identification of conserved TFBSs by improving the alignment accuracy of TFBS families within orthologous DNA sequences. Functional validation of the Gfi1b + 13 enhancer reveals that ConBind identifies additional functionally important ETS binding sites that were missed by all other tested alignment tools. In addition to the analysis of known regulatory regions, our web tool is useful for the analysis of TFBSs on so far unknown DNA regions identified through ChIP-sequencing. PMID:26721389

  13. Fibronectin type III5 repeat contains a novel cell adhesion sequence, KLDAPT, which binds activated alpha4beta1 and alpha4beta7 integrins.

    PubMed

    Moyano, J V; Carnemolla, B; Domínguez-Jiménez, C; García-Gila, M; Albar, J P; Sánchez-Aparicio, P; Leprini, A; Querzé, G; Zardi, L; Garcia-Pardo, A

    1997-10-03

    The region of fibronectin encompassing type III repeats 4-6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact with cells as already shown for other heparin binding domains of fibronectin. A computer search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT located in FN-III5. A synthetic peptide containing this sequence induced lymphoid cell adhesion upon treatment with the activating anti-beta1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating that KLDAPT was binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5) also mediated adhesion of TS2/16/Mn2+-treated cells while the FN-III6 fragment did not. Soluble KLDAPT peptide inhibited cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and VCAM-1, two previously known ligands for alpha4beta1 integrin. KLDAPT also competed with the binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated alpha4beta1. Furthermore, mAbs anti-alpha4 and anti-alpha4beta7, but not mAbs to other integrins, inhibited cell adhesion to FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5 repeat and show that KLDAPT is a novel fibronectin ligand for activated alpha4 integrins.

  14. The LIM motif defines a specific zinc-binding protein domain.

    PubMed

    Michelsen, J W; Schmeichel, K L; Beckerle, M C; Winge, D R

    1993-05-15

    The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-11, Is1-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are reminiscent of a metal-binding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) and from a bacterial expression system and observed that cCRP is a specific Zn-binding metalloprotein. Four Zn(II) ions are maximally bound to cCRP, consistent with the idea that each LIM domain coordinates two metal ions. From spectroscopic studies of Co(II)- and 113Cd(II)-substituted cCRP, we determined that each metal ion is tetrahedrally coordinated with cysteinyl sulfurs dominating the ligand types. One metal site within each LIM motif has tetrathiolate (S4) coordination, the second site may either be S4 or S3N1. The LIM motif represents another example of a specific Zn-binding protein sequence.

  15. Fibronectin Binding to the Treponema pallidum Adhesin Protein Fragment rTp0483 on Functionalized Self-Assembled Monolayers

    PubMed Central

    Dickerson, Matthew T.; Abney, Morgan B.; Cameron, Caroline E.; Knecht, Marc; Bachas, Leonidas G.; Anderson, Kimberly W.

    2012-01-01

    Past work has shown that Treponema pallidum, the causative agent of syphilis, binds host fibronectin (FN). FN and other host proteins are believed to bind to rare outer membrane proteins (OMPs) of T. pallidum, and it is postulated that this interaction may facilitate cell attachment and mask antigenic targets on the surface. This research seeks to prepare a surface capable of mimicking the FN binding ability of T. pallidum in order to investigate the impact of FN binding with adsorbed Tp0483 on the host response to the surface. By understanding this interaction it may be possible to develop more effective treatments for infection and possibly mimic the stealth properties of the bacteria. Functionalized self-assembled monolayers (SAMs) on0 gold were used to investigate rTp0483 and FN adsorption. Using a quartz crystal microbalance (QCM) rTp0483 adsorption and subsequent FN adsorption onto rTp0483 was determined to be higher on negatively charged carboxylate-terminated self-assembled monolayers (−COO− SAMs) compared to the other surfaces analyzed. Kinetic analysis of rTp0483 adsorption using surface plasmon resonance (SPR) supported this finding. Kinetic analysis of FN adsorption using SPR revealed a multi-step event, where the concentration of immobilized rTp0483 plays a role in FN binding. An examination of relative QCM dissipation energy compared to the shift in frequency showed a correlation between the physical properties of adsorbed rTp0483 and SAM surface chemistry. In addition, AFM images of rTp0483 on selected SAMs illustrated a preference of rTp0483 to bind as aggregates. Adsorption on −COO− SAMs was more uniform across the surface, which may help further explain why FN bound more strongly. rTp0483 antibody studies suggested the involvement of amino acids 274–289 and 316–333 in binding between rTp0483 to FN, while a peptide blocking study only showed inhibition of binding with amino acids 316–333. Finally, surface adsorbed rTp0483 with FN

  16. Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif.

    PubMed Central

    Kudrycki, K; Stein-Izsak, C; Behn, C; Grillo, M; Akeson, R; Margolis, F L

    1993-01-01

    We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP. Images PMID:8474458

  17. Molecular diversity of LysM carbohydrate-binding motifs in fungi.

    PubMed

    Akcapinar, Gunseli Bayram; Kappel, Lisa; Sezerman, Osman Ugur; Seidl-Seiboth, Verena

    2015-05-01

    LysM motifs are carbohydrate-binding modules found in prokaryotes and eukaryotes. They bind to N-acetylglucosamine-containing carbohydrates, such as chitin, chitio-oligosaccharides and peptidoglycan. In this review, we summarize the features of the protein architecture of LysM-containing proteins in fungi and discuss their so far known biochemical properties, transcriptional profiles and biological functions. Further, based on data from evolutionary analyses and consensus pattern profiling of fungal LysM motifs, we show that they can be classified into a fungal-specific group and a fungal/bacterial group. This facilitates the classification and selection of further LysM proteins for detailed analyses and will contribute to widening our understanding of the functional spectrum of this protein family in fungi. Fungal LysM motifs are predominantly found in subgroup C chitinases and in LysM effector proteins, which are secreted proteins with LysM motifs but no catalytic domains. In enzymes, LysM motifs mediate the attachment to insoluble carbon sources. In plants, receptors containing LysM motifs are responsible for the perception of chitin-oligosaccharides and are involved in beneficial symbiotic interactions between plants and bacteria or fungi, as well as plant defence responses. In plant pathogenic fungi, LysM effector proteins have already been shown to have important functions in the dampening of host defence responses as well as protective functions of fungal hyphae against chitinases. However, the large number and diversity of proteins with LysM motifs that are being unravelled in fungal genome sequencing projects suggest that the functional repertoire of LysM effector proteins in fungi is only partially discovered so far.

  18. Sequence-motif Detection of NAD(P)-binding Proteins: Discovery of a Unique Antibacterial Drug Target

    NASA Astrophysics Data System (ADS)

    Hua, Yun Hao; Wu, Chih Yuan; Sargsyan, Karen; Lim, Carmay

    2014-09-01

    Many enzymes use nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (NAD(P)) as essential coenzymes. These enzymes often do not share significant sequence identity and cannot be easily detected by sequence homology. Previously, we determined all distinct locally conserved pyrophosphate-binding structures (3d motifs) from NAD(P)-bound protein structures, from which 1d sequence motifs were derived. Here, we aim to establish the precision of these 3d and 1d motifs to annotate NAD(P)-binding proteins. We show that the pyrophosphate-binding 3d motifs are characteristic of NAD(P)-binding proteins, as they are rarely found in nonNAD(P)-binding proteins. Furthermore, several 1d motifs could distinguish between proteins that bind only NAD and those that bind only NADP. They could also distinguish between NAD(P)-binding proteins from nonNAD(P)-binding ones. Interestingly, one of the pyrophosphate-binding 3d and corresponding 1d motifs was found only in enoyl-acyl carrier protein reductases, which are enzymes essential for bacterial fatty acid biosynthesis. This unique 3d motif serves as an attractive novel drug target, as it is conserved across many bacterial species and is not found in human proteins.

  19. An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

    PubMed Central

    Sanchez-Alberola, Neus; Campoy, Susana; Emerson, David; Barbé, Jordi

    2015-01-01

    ABSTRACT The SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In the Betaproteobacteria, LexA is known to target a palindromic sequence with the consensus sequence CTGT-N8-ACAG. We report the characterization of a LexA regulon in the iron-oxidizing betaproteobacterium Sideroxydans lithotrophicus. In silico and in vitro analyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of the Bacillus subtilis LexA-binding motif. We confirm that the closely related Gallionella capsiferriformans shares the same LexA-binding motif, and in silico analyses indicate that this motif is also conserved in the Nitrosomonadales and the Methylophilales. Phylogenetic analysis of LexA and the alpha subunit of DNA polymerase III (DnaE) reveal that the organisms harboring this noncanonical LexA form a compact taxonomic cluster within the Betaproteobacteria. However, their lexA gene is unrelated to the standard Betaproteobacteria lexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon of S. lithotrophicus is comparable in size and function to that of many other Betaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA protein. Analysis of the DNA-binding domain of S. lithotrophicus LexA reveals little sequence similarity with that of other LexA proteins targeting similar binding motifs, suggesting that network structure may limit site evolution or that structural constrains make the B. subtilis-type motif an optimal interface for multiple LexA sequences. IMPORTANCE Understanding the evolution of transcriptional systems enables us to address important questions in microbiology, such as the emergence and transfer potential of different regulatory systems to regulate virulence or

  20. Localization of the domains of the Haemophilus ducreyi trimeric autotransporter DsrA involved in serum resistance and binding to the extracellular matrix proteins fibronectin and vitronectin.

    PubMed

    Leduc, Isabelle; Olsen, Bonnie; Elkins, Christopher

    2009-02-01

    Resisting the bactericidal activity of naturally occurring antibodies and complement of normal human serum is an important element in the evasion of innate immunity by bacteria. In the gram-negative mucosal pathogen Haemophilus ducreyi, serum resistance is mediated primarily by the trimeric autotransporter DsrA. DsrA also functions as an adhesin for the extracellular matrix proteins fibronectin and vitronectin and mediates attachment of H. ducreyi to keratinocytes. We sought to determine the domain(s) of the 236-residue DsrA protein required for serum resistance and extracellular matrix protein binding. A 140-amino-acid truncated protein containing only the C-terminal portion of the passenger domain and the entire translocator domain of DsrA exhibited binding to fibronectin and vitronectin and conferred serum resistance to an H. ducreyi serum-sensitive strain. A shorter DsrA construct consisting of only 128 amino acids was unable to bind to extracellular matrix proteins but was serum resistant. We concluded that neither fibronectin binding nor vitronectin binding is required for high-level serum resistance in H. ducreyi.

  1. Localization of the Domains of the Haemophilus ducreyi Trimeric Autotransporter DsrA Involved in Serum Resistance and Binding to the Extracellular Matrix Proteins Fibronectin and Vitronectin▿

    PubMed Central

    Leduc, Isabelle; Olsen, Bonnie; Elkins, Christopher

    2009-01-01

    Resisting the bactericidal activity of naturally occurring antibodies and complement of normal human serum is an important element in the evasion of innate immunity by bacteria. In the gram-negative mucosal pathogen Haemophilus ducreyi, serum resistance is mediated primarily by the trimeric autotransporter DsrA. DsrA also functions as an adhesin for the extracellular matrix proteins fibronectin and vitronectin and mediates attachment of H. ducreyi to keratinocytes. We sought to determine the domain(s) of the 236-residue DsrA protein required for serum resistance and extracellular matrix protein binding. A 140-amino-acid truncated protein containing only the C-terminal portion of the passenger domain and the entire translocator domain of DsrA exhibited binding to fibronectin and vitronectin and conferred serum resistance to an H. ducreyi serum-sensitive strain. A shorter DsrA construct consisting of only 128 amino acids was unable to bind to extracellular matrix proteins but was serum resistant. We concluded that neither fibronectin binding nor vitronectin binding is required for high-level serum resistance in H. ducreyi. PMID:19015257

  2. Evidence for a Structural Motif in Toxins and Interleukin-2 That May Be Responsible for Binding to Endothelial Cells and Initiating Vascular Leak Syndrome

    NASA Astrophysics Data System (ADS)

    Baluna, Roxana; Rizo, Josep; Gordon, Brian E.; Ghetie, Victor; Vitetta, Ellen S.

    1999-03-01

    The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

  3. Fibrillin Assembly Requires Fibronectin

    PubMed Central

    Sabatier, Laetitia; Chen, Daliang; Fagotto-Kaufmann, Christine; Hubmacher, Dirk; McKee, Marc D.; Annis, Douglas S.; Mosher, Deane F.

    2009-01-01

    Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and nonelastic extracellular matrices. Proper assembly mechanisms are central to the formation and function of these microfibrils, and their properties are often compromised in pathological circumstances such as in Marfan syndrome and in other fibrillinopathies. Here, we have used human dermal fibroblasts to analyze the assembly of fibrillin-1 in dependence of other matrix-forming proteins. siRNA knockdown experiments demonstrated that the assembly of fibrillin-1 is strictly dependent on the presence of extracellular fibronectin fibrils. Immunolabeling performed at the light and electron microscopic level showed colocalization of fibrillin-1 with fibronectin fibrils at the early stages of the assembly process. Protein-binding assays demonstrated interactions of fibronectin with a C-terminal region of fibrillin-1, -2, and -3 and with an N-terminal region of fibrillin-1. The C-terminal half of fibrillin-2 and -3 had propensities to multimerize, as has been previously shown for fibrillin-1. The C-terminal of all three fibrillins interacted strongly with fibronectin as multimers, but not as monomers. Mapping studies revealed that the major binding interaction between fibrillins and fibronectin involves the collagen/gelatin-binding region between domains FNI6 and FNI9. PMID:19037100

  4. Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation

    PubMed Central

    Sun, Chaomin; Querol-Audí, Jordi; Mortimer, Stefanie A.; Arias-Palomo, Ernesto; Doudna, Jennifer A.; Nogales, Eva; Cate, Jamie H. D.

    2013-01-01

    The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome. PMID:23766293

  5. Discovery of widespread GTP-binding motifs in genomic DNA and RNA.

    PubMed

    Curtis, Edward A; Liu, David R

    2013-04-18

    Biological RNAs that bind small molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genome-encoded RNA fragments for naturally occurring GTP aptamers. Several aptamer classes were identified, including one (the "G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ~75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (~300 μM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.

  6. A Common Structural Motif in the Binding of Virulence Factors to Bacterial Secretion Chaperones

    SciTech Connect

    Lilic,M.; Vujanac, M.; Stebbins, C.

    2006-01-01

    Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.

  7. Identification of the RGG Box Motif in Shadoo: RNA-Binding and Signaling Roles?

    PubMed Central

    Corley, Susan M.; Gready, Jill E.

    2008-01-01

    Using comparative genomics and in-silico analyses, we previously identified a new member of the prion-protein (PrP) family, the gene SPRN, encoding the protein Shadoo (Sho), and suggested its functions might overlap with those of PrP. Extended bioinformatics and conceptual biology studies to elucidate Sho’s functions now reveal Sho has a conserved RGG-box motif, a well-known RNA-binding motif characterized in proteins such as FragileX Mental Retardation Protein. We report a systematic comparative analysis of RGG-box containing proteins which highlights the motif’s functional versatility and supports the suggestion that Sho plays a dual role in cell signaling and RNA binding in brain. These findings provide a further link to PrP, which has well-characterized RNA-binding properties. PMID:19812790

  8. Novel DNA motif binding activity observed in vivo with an estrogen receptor α mutant mouse.

    PubMed

    Hewitt, Sylvia C; Li, Leping; Grimm, Sara A; Winuthayanon, Wipawee; Hamilton, Katherine J; Pockette, Brianna; Rubel, Cory A; Pedersen, Lars C; Fargo, David; Lanz, Rainer B; DeMayo, Francesco J; Schütz, Günther; Korach, Kenneth S

    2014-06-01

    Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

  9. Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse

    PubMed Central

    Li, Leping; Grimm, Sara A.; Winuthayanon, Wipawee; Hamilton, Katherine J.; Pockette, Brianna; Rubel, Cory A.; Pedersen, Lars C.; Fargo, David; Lanz, Rainer B.; DeMayo, Francesco J.; Schütz, Günther; Korach, Kenneth S.

    2014-01-01

    Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as “tethering.” Evidence for tethering is based on in vitro studies and a widely used “KIKO” mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the “EAAE” ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null–like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo. PMID:24713037

  10. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

    PubMed

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  11. Alternative peptide binding motifs of Qa-2 class Ib molecules define rules for binding of self and nonself peptides.

    PubMed

    Tabaczewski, P; Chiang, E; Henson, M; Stroynowski, I

    1997-09-15

    Studies of naturally processed peptides eluted from membrane-bound and soluble isoforms of murine class Ib Qa-2 molecules determined several features of these ligands, such as the conserved nonameric length and the preferred usage of specific residues at four to six of nine peptide positions. The structural information derived from these studies proved insufficient to distinguish between two interpretations: 1) that Qa-2 are peptide receptors of higher stringency than ordinary class I molecules, and 2) that Qa-2 molecules, like classical class I Ags, bind diverse arrays of peptides. We have addressed this issue by a systematic analysis of peptide residues involved in the binding of membrane-bound Qa-2 molecule, MQ9b. The optimal binding of synthetic peptides in vitro occurs at neutral pH. Two dominant anchors are required for peptide binding to MQ9b: His at position 7 and a hydrophobic residue, Leu, Ile, or Phe, at position 9. In addition, one or two auxiliary anchors participate in binding. The identity and the position of the auxiliary anchors differ from peptide to peptide, suggesting that the binding motifs defined from pool sequencing are composed of many superimposed alternative motifs present in individual peptides. The number of anchors used by Qa-2 peptides is similar to that found in ligands of classical class I Ags. Consequently, the Qa-2 are predicted to bind large repertoires of self and nonself peptides. In support of this interpretation we demonstrate that MQ9b binds strongly 5 of 17 motif-positive, pathogen-derived synthetic peptides.

  12. A threonine turnstile defines a dynamic amphiphilic binding motif in the AAA ATPase p97 allosteric binding site.

    PubMed

    Burnett, James C; Lim, Chaemin; Peyser, Brian D; Samankumara, Lalith P; Kovaliov, Marina; Colombo, Raffaele; Bulfer, Stacie L; LaPorte, Matthew G; Hermone, Ann R; McGrath, Connor F; Arkin, Michelle R; Gussio, Rick; Huryn, Donna M; Wipf, Peter

    2017-03-29

    The turnstile motion of two neighboring threonines sets up a dynamic side chain interplay that can accommodate both polar and apolar ligands in a small molecule allosteric protein binding site. A computational model based on SAR data and both X-ray and cryo-EM structures of the AAA ATPase p97 was used to analyze the effects of paired threonines at the inhibitor site. Specifically, the Thr side chain hydroxyl groups form a hydrogen bonding network that readily accommodates small, highly polar ligand substituents. Conversely, diametric rotation of the χ1 torsion by 150-180° orients the side chain β-methyl groups into the binding cleft, creating a hydrophobic pocket that can accommodate small, apolar substituents. This motif was found to be critical for rationalizing the affinities of a structurally focused set of inhibitors of p97 covering a > 2000-fold variation in potencies, with a preference for either small-highly polar or small-apolar groups. The threonine turnstile motif was further validated by a PDB search that identified analogous binding modes in ligand interactions in PKB, as well as by an analysis of NMR structures demonstrating additional gear-like interactions between adjacent Thr pairs. Combined, these data suggest that the threonine turnstile motif may be a general feature of interest in protein binding pockets.

  13. A monoclonal antibody inhibits gelatinase B/MMP-9 by selective binding to part of the catalytic domain and not to the fibronectin or zinc binding domains.

    PubMed

    Martens, Erik; Leyssen, An; Van Aelst, Ilse; Fiten, Pierre; Piccard, Helene; Hu, Jialiang; Descamps, Francis J; Van den Steen, Philippe E; Proost, Paul; Van Damme, Jo; Liuzzi, Grazia Maria; Riccio, Paolo; Polverini, Eugenia; Opdenakker, Ghislain

    2007-02-01

    Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a multidomain enzyme functioning in acute and chronic inflammatory and neoplastic diseases. It belongs to a family of more than 20 related zinc proteinases. Therefore, the discovery and the definition of the action mechanism of selective MMP inhibitors form the basis for future therapeutics. The monoclonal antibody REGA-3G12 is a most selective inhibitor of human gelatinase B. REGA-3G12 was found to recognize the aminoterminal part and not the carboxyterminal O-glycosylated and hemopexin protein domains. A variant of gelatinase B, lacking the two carboxyterminal domains, was expressed in insect cells and fragmented with purified proteinases. The fragments were probed by one- and two-dimensional Western blot and immunoprecipitation experiments with REGA-3G12 to map the interactions between the antibody and the enzyme. The interaction unit was identified by Edman degradation analysis as the glycosylated segment from Trp(116) to Lys(214) of gelatinase B. The sequence of this segment was analysed by hydrophobicity/hydrophilicity, accessibility and flexibility profiling. Four hydrophilic peptides were chemically synthesized and used in binding and competition assays. The peptide Gly(171)-Leu(187) in molar excess inhibited partially the binding of MMP-9 to REGA-3G12 and thus refines the structure of the conformational binding site. These results define part of the catalytic domain of gelatinase B/MMP-9, and not the zinc-binding or fibronectin domains, as target for the development of selective inhibitors.

  14. Blockade of very late antigen-4 integrin binding to fibronectin with connecting segment-1 peptide reduces accelerated coronary arteriopathy in rabbit cardiac allografts.

    PubMed Central

    Molossi, S; Elices, M; Arrhenius, T; Diaz, R; Coulber, C; Rabinovitch, M

    1995-01-01

    Graft arteriopathy, a leading cause of cardiac allograft failure, is associated with increased intimal smooth muscle cells, inflammatory cells, and accumulation of extracellular matrix. We hypothesized that cellular fibronectin plays a pivotal role in the progression of the allograft arteriopathy by directing the transendothelial trafficking of inflammatory cells through interaction of the connecting segment-1 (CS1) motif with the very late antigen-4 (VLA-4) integrin, and tested this in vivo using a blocking peptide. Cholesterol-fed rabbits underwent heterotopic cardiac transplantation without immunosuppression. The treatment group (n = 7) received a synthetic CS1 peptide (1 mg/kg per d, subcutaneously), and the controls (n = 7) received an inactive peptide (1 mg/kg per d, subcutaneously). At 7-8 d after transplantation, hearts were harvested and sectioned for morphometric analysis and immunohistochemical studies. We observed a > 50% decrease in the incidence (P < 0.001) and severity (P < 0.001) of donor coronary artery intimal thickening in the CS1-treated compared with the control group. These findings correlated with reduced infiltration of T cells (P < 0.05), a trend toward decreased expression of adhesion molecules (P < 0.06), and less accumulation of fibronectin (P < 0.03). Our data suggest that the VLA-4-fibronectin interaction is critical to the progression of the allograft arteriopathy by perpetuating the immune-inflammatory response in the vessel wall. Images PMID:7539456

  15. Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

    PubMed

    Dalrymple, Brian P; Kongsuwan, Kritaya; Wijffels, Gene

    2007-01-01

    Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

  16. AliBiMotif: integrating alignment and biclustering to unravel transcription factor binding sites in DNA sequences.

    PubMed

    Gonçalves, Joana P; Moreau, Yves; Madeira, Sara C

    2012-01-01

    Transcription Factors (TFs) control transcription by binding to specific sites in the promoter regions of the target genes, which can be modelled by structured motifs. In this paper we propose AliBiMotif, a method combining sequence alignment and a biclustering approach based on efficient string matching techniques using suffix trees to unravel approximately conserved sets of blocks (structured motifs) while straightforwardly disregarding non-conserved stretches in-between. The ability to ignore the width of non-conserved regions is a major advantage of the proposed method over other motif finders, as the lengths of the binding sites are usually easier to estimate than the separating distances.

  17. A small fibronectin-mimicking protein from bacteria induces cell spreading and focal adhesion formation.

    PubMed

    Tegtmeyer, Nicole; Hartig, Roland; Delahay, Robin M; Rohde, Manfred; Brandt, Sabine; Conradi, Jens; Takahashi, Seiichiro; Smolka, Adam J; Sewald, Norbert; Backert, Steffen

    2010-07-23

    Fibronectin, a 250-kDa eukaryotic extracellular matrix protein containing an RGD motif plays crucial roles in cell-cell communication, development, tissue homeostasis, and disease development. The highly complex fibrillar fibronectin meshwork orchestrates the functions of other extracellular matrix proteins, promoting cell adhesion, migration, and intracellular signaling. Here, we demonstrate that CagL, a 26-kDa protein of the gastric pathogen and type I carcinogen Helicobacter pylori, mimics fibronectin in various cellular functions. Like fibronectin, CagL contains a RGD motif and is located on the surface of the bacterial type IV secretion pili as previously shown. CagL binds to the integrin receptor alpha(5)beta(1) and mediates the injection of virulence factors into host target cells. We show that purified CagL alone can directly trigger intracellular signaling pathways upon contact with mammalian cells and can complement the spreading defect of fibronectin(-/-) knock-out cells in vitro. During interaction with various human and mouse cell lines, CagL mimics fibronectin in triggering cell spreading, focal adhesion formation, and activation of several tyrosine kinases in an RGD-dependent manner. Among the activated factors are the nonreceptor tyrosine kinases focal adhesion kinase and Src but also the epidermal growth factor receptor and epidermal growth factor receptor family member Her3/ErbB3. Interestingly, fibronectin activates a similar range of tyrosine kinases but not Her3/ErbB3. These findings suggest that the bacterial protein CagL not only exhibits functional mimicry with fibronectin but is also capable of activating fibronectin-independent signaling events. We thus postulate that CagL may contribute directly to H. pylori pathogenesis by promoting aberrant signaling cross-talk within host cells.

  18. Identification of sequence–structure RNA binding motifs for SELEX-derived aptamers

    PubMed Central

    Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E.; Przytycka, Teresa M.

    2012-01-01

    Motivation: Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. Results: To close this gap we developed, Aptamotif, a computational method for the identification of sequence–structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process. Contact: przytyck@ncbi.nlm.nih.gov, Zuben.Sauna@fda.hhs.gov PMID:22689764

  19. Numb directs the subcellular localization of EAAT3 through binding the YxNxxF motif.

    PubMed

    Su, Jin-Feng; Wei, Jian; Li, Pei-Shan; Miao, Hong-Hua; Ma, Yong-Chao; Qu, Yu-Xiu; Xu, Jie; Qin, Jie; Li, Bo-Liang; Song, Bao-Liang; Xu, Zheng-Ping; Luo, Jie

    2016-08-15

    Excitatory amino acid transporter type 3 (EAAT3, also known as SLC1A1) is a high-affinity, Na(+)-dependent glutamate carrier that localizes primarily within the cell and at the apical plasma membrane. Although previous studies have reported proteins and sequence regions involved in EAAT3 trafficking, the detailed molecular mechanism by which EAAT3 is distributed to the correct location still remains elusive. Here, we identify that the YVNGGF sequence in the C-terminus of EAAT3 is responsible for its intracellular localization and apical sorting in rat hepatoma cells CRL1601 and Madin-Darby canine kidney (MDCK) cells, respectively. We further demonstrate that Numb, a clathrin adaptor protein, directly binds the YVNGGF motif and regulates the localization of EAAT3. Mutation of Y503, N505 and F508 within the YVNGGF motif to alanine residues or silencing Numb by use of small interfering RNA (siRNA) results in the aberrant localization of EAAT3. Moreover, both Numb and the YVNGGF motif mediate EAAT3 endocytosis in CRL1601 cells. In summary, our study suggests that Numb is a pivotal adaptor protein that mediates the subcellular localization of EAAT3 through binding the YxNxxF (where x stands for any amino acid) motif.

  20. ATtRACT-a database of RNA-binding proteins and associated motifs.

    PubMed

    Giudice, Girolamo; Sánchez-Cabo, Fátima; Torroja, Carlos; Lara-Pezzi, Enrique

    2016-01-01

    RNA-binding proteins (RBPs) play a crucial role in key cellular processes, including RNA transport, splicing, polyadenylation and stability. Understanding the interaction between RBPs and RNA is key to improve our knowledge of RNA processing, localization and regulation in a global manner. Despite advances in recent years, a unified non-redundant resource that includes information on experimentally validated motifs, RBPs and integrated tools to exploit this information is lacking. Here, we developed a database named ATtRACT (available athttp://attract.cnic.es) that compiles information on 370 RBPs and 1583 RBP consensus binding motifs, 192 of which are not present in any other database. To populate ATtRACT we (i) extracted and hand-curated experimentally validated data from CISBP-RNA, SpliceAid-F, RBPDB databases, (ii) integrated and updated the unavailable ASD database and (iii) extracted information from Protein-RNA complexes present in Protein Data Bank database through computational analyses. ATtRACT provides also efficient algorithms to search a specific motif and scan one or more RNA sequences at a time. It also allows discoveringde novomotifs enriched in a set of related sequences and compare them with the motifs included in the database.Database URL:http:// attract. cnic. es.

  1. Binding of the Grb2 SH2 domain to phosphotyrosine motifs does not change the affinity of its SH3 domains for Sos proline-rich motifs.

    PubMed

    Cussac, D; Frech, M; Chardin, P

    1994-09-01

    Phosphotyrosine peptide binding to Grb2 induces tryptophan fluorescence changes in the Src homology 2 (SH2) domain. Affinities are in the nanomolar range, the Shc peptide having the highest affinity, followed by peptides mimicking Grb2 binding sites on EGF and HGF receptors, the putative sites on insulin and IGF-1 receptors having much lower affinities. Proline-rich peptide binding to the SH3 domains induces fluorescence changes mainly in the C-terminal SH3. Affinities are in the micromolar range, the highest affinity peptides mimicking the first proline-rich motif of the Sos C-terminus. Additional residues before this PVPPPVPP motif provide a minor contribution to the binding, but the two residues after this motif are important and may contribute to specificity. The affinity of each SH3 for each proline-rich motif is too low to account for the high stability of the Grb2-Sos complex, suggesting that Grb2 recognizes other structural features in the Sos C-terminus. Binding of a phosphotyrosine peptide to the SH2 has no effect on the SH3s. Thus the binding of Grb2 to a receptor or to an associated protein phosphorylated on tyrosines is unlikely to activate the exchange factor activity of Sos through a conformational change transmitted from the SH2 to the SH3 domains.

  2. Role of Ets-1 in fibronectin-derived heparin-binding domain polypeptides alleviating melanoma cell invasiveness and chemoresistance.

    PubMed

    Tang, Nanhong; Wang, Xiaoqian; Huang, Tao; Wu, Yong; Chen, Yuanzhong

    2014-07-01

    In this study, we observed that rhFNHN29 and rhFNHC36, two recombinant heparin-binding domain polypeptides of fibronectin, suppressed adhesion and invasion of B16F10 and A375 melanoma cells mediated by integrin αv and α2 in a dose-dependent manner. Combined with low-concentration epirubicin (EPI), rhFNHN29 or rhFNHC36 exhibited a synergistic inhibition on the viability and metastasis of B16F10 cells. Moreover, in the presence of high-concentration rhFNHN29 or rhFNHC36, the Ets-1 activity and the expression of p-FAK, p-Erk1/2 and Ets-1 were notably downregulated in B16F10 cells. Ets-1 is one of the central regulatory links for rhFNHN29 and rhFNHC36 to suppress the adhesion and invasion of melanoma cells. Combining rhFNHN29 or rhFNHC36 with EPI may be a good way to alleviate invasiveness or chemoresistance in melanoma.

  3. Polymorphisms in Fibronectin Binding Proteins A and B among Staphylococcus aureus Bloodstream Isolates Are Not Associated with Arthroplasty Infection

    PubMed Central

    Sharma-Kuinkel, Batu; Park, Lawrence P.; Rude, Thomas H.; Ruffin, Felicia; Hos, Nina J.; Seifert, Harald; Rieg, Siegbert; Kern, Winfried V.; Lower, Steven K.; Fowler, Vance G.; Kaasch, Achim J.

    2015-01-01

    Background Nonsynonymous single nucleotide polymorphisms (SNPs) in fibronectin binding protein A (fnbA) of Staphylococcus aureus are associated with cardiac device infections. However, the role of fnbA SNPs in S. aureus arthroplasty infection is unknown. Methods Bloodstream S. aureus isolates from a derivation cohort of patients at a single U.S. medical center with S. aureus bacteremia (SAB) and prosthetic hip or knee arthroplasties that were infected (PJI, n = 27) or uninfected (PJU, n = 43) underwent sequencing of fnbA and fnbB. A validation cohort of S. aureus bloodstream PJI (n = 12) and PJU (n = 58) isolates from Germany also underwent fnbA and fnbB sequencing. Results Overall, none of the individual fnbA or fnbB SNPs were significantly associated with the PJI or PJU clinical groups within the derivation cohort. Similarly, none of the individual fnbA or fnbB SNPs were associated with PJI or PJU when the analysis was restricted to patients with either early SAB (i.e., bacteremia occurring <1 year after placement or manipulation of prostheses) or late SAB (i.e., bacteremia >1 year after placement or manipulation of prostheses). Conclusions In contrast to cardiac device infections, there is no association between nonsynonymous SNPs in fnbA or fnbB of bloodstream S. aureus isolates and arthroplasty infection. These results suggest that initial steps leading to S. aureus infection of cardiovascular and orthopedic prostheses may arise by distinct processes. PMID:26606522

  4. Biochemical characterization of PE_PGRS61 family protein of Mycobacterium tuberculosis H37Rv reveals the binding ability to fibronectin

    PubMed Central

    Monu; Meena, Laxman S

    2016-01-01

    Objective(s): The periodic binding of protein expressed by Mycobacterium tuberculosis H37Rv with the host cell receptor molecules i.e. fibronectin (Fn) is gaining significance because of its adhesive properties. The genome sequencing of M. tuberculosis H37Rv revealed that the proline-glutamic (PE) proteins contain polymorphic GC-rich repetitive sequences (PGRS) which have clinical importance in pathogenesis events when the host encounters M. tuberculosis H37Rv. The functional parts of PE_PGRS family proteins, have not been extensively studied in tuberculosis biology. Materials and Methods: Fibronectin (10 ng and 20 ng) were used for FnBP assay and its enzymatic activities were observed by using various protein concentrations. Results: Therefore, in the present work, we cloned, expressed, purified and identified a novel PE_PGRS61 (Rv3653) family protein in M. tuberculosis H37Rv. Our experiment, observation suggested that at particular concentrations of 10 ng and 20 ng of Fn exhibits optimum binding to the purified Fibronectin Binding Protein (FnBP), a PE_PGRS61 family protein at 0.20 μg and 0.25 μg concentrations, respectively. Moreover, for better understanding the computational analysis, the B-cell and T-cell epitopes prediction prospect some amino acid propensity scales with hydrophilicity and antigenic variation index at their respective locations. Conclusion: Thus, the current findings provide an opportunity to illuminate the functions of PE_PGRS61 family protein. So, in this point of view, it could be useful to develop a novel therapeutic approach or diagnostic pipeline through targeting these fibronectin binding protein (FnBP) expressing genes. PMID:27872707

  5. ICAP-1, a Novel β1 Integrin Cytoplasmic Domain–associated Protein, Binds to a Conserved and Functionally Important NPXY Sequence Motif of β1 Integrin

    PubMed Central

    Chang, David D.; Wong, Carol; Smith, Healy; Liu, Jenny

    1997-01-01

    The cytoplasmic domains of integrins are essential for cell adhesion. We report identification of a novel protein, ICAP-1 (integrin cytoplasmic domain– associated protein-1), which binds to the β1 integrin cytoplasmic domain. The interaction between ICAP-1 and β1 integrins is highly specific, as demonstrated by the lack of interaction between ICAP-1 and the cytoplasmic domains of other β integrins, and requires a conserved and functionally important NPXY sequence motif found in the COOH-terminal region of the β1 integrin cytoplasmic domain. Mutational studies reveal that Asn and Tyr of the NPXY motif and a Val residue located NH2-terminal to this motif are critical for the ICAP-1 binding. Two isoforms of ICAP-1, a 200–amino acid protein (ICAP-1α) and a shorter 150–amino acid protein (ICAP-1β), derived from alternatively spliced mRNA, are expressed in most cells. ICAP-1α is a phosphoprotein and the extent of its phosphorylation is regulated by the cell–matrix interaction. First, an enhancement of ICAP-1α phosphorylation is observed when cells were plated on fibronectin-coated but not on nonspecific poly-l-lysine–coated surface. Second, the expression of a constitutively activated RhoA protein that disrupts the cell–matrix interaction results in dephosphorylation of ICAP-1α. The regulation of ICAP-1α phosphorylation by the cell–matrix interaction suggests an important role of ICAP-1 during integrin-dependent cell adhesion. PMID:9281591

  6. Transcriptional regulation of adhesive properties of Bacillus subtilis to extracellular matrix proteins through the fibronectin-binding protein YloA.

    PubMed

    Rodriguez Ayala, Facundo; Bauman, Carlos; Bartolini, Marco; Saball, Ester; Salvarrey, Marcela; Leñini, Cecilia; Cogliati, Sebastián; Strauch, Mark; Grau, Roberto

    2017-03-13

    Bacterial adherence to extracellular matrix proteins (ECMp) plays important roles during host-pathogen interaction, however its genetic regulation remains poorly understood. yloA of the model bacterium Bacillus subtilis shows high homology to genes encoding fibronectin-binding proteins of Gram-positive pathogens. Here, we characterized the regulatory network of YloA-dependent adhesive properties of the probiotic B. subtilis natto (Bsn). YloA-proficient, but not YloA-deficient, Bsn specifically bound to ECMp in a concentration-dependent manner and were proficient in biofilm formation. yloA expression showed a continuous increase in activity during the growth phase and decreased during the stationary phase. The transcription factors AbrB and DegU downregulated yloA expression during the logarithmic and stationary growth phases respectively. Analysis of the yloA promoter region revealed the presence of AT-rich direct and inverted repeats previously reported to function as DegU-recognized binding sites. In spo0A cells, yloA expression was completely turned off because of upregulation of AbrB throughout growth. Accordingly, DNase I footprinting analysis confirmed that AbrB bound to the promoter region of yloA. Interestingly, Bsn bound fibronectin with higher affinity, lower Kd, than several bacterial pathogens and competitively excluded them from binding to immobilized-fibronectin, a finding that might be important for the anti-infective properties of B. subtilis and its relatives.

  7. Staphylococcus aureus Fibronectin Binding Proteins Are Essential for Internalization by Osteoblasts but Do Not Account for Differences in Intracellular Levels of Bacteria

    PubMed Central

    Ahmed, Saddif; Meghji, Sajeda; Williams, Rachel J.; Henderson, Brian; Brock, Jeremy H.; Nair, Sean P.

    2001-01-01

    Staphylococcus aureus is a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake of S. aureus by osteoblasts. An S. aureus septic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains of S. aureus blocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains of S. aureus are internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process. PMID:11292701

  8. Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

    NASA Astrophysics Data System (ADS)

    Qian, Long; Kussell, Edo

    2016-10-01

    The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

  9. Transcription Factor Binding Site Positioning in Yeast: Proximal Promoter Motifs Characterize TATA-Less Promoters

    PubMed Central

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of ‘proximal promoter motifs’ (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters. PMID:21931670

  10. DNA familial binding profiles made easy: comparison of various motif alignment and clustering strategies.

    PubMed

    Mahony, Shaun; Auron, Philip E; Benos, Panayiotis V

    2007-03-30

    Transcription factor (TF) proteins recognize a small number of DNA sequences with high specificity and control the expression of neighbouring genes. The evolution of TF binding preference has been the subject of a number of recent studies, in which generalized binding profiles have been introduced and used to improve the prediction of new target sites. Generalized profiles are generated by aligning and merging the individual profiles of related TFs. However, the distance metrics and alignment algorithms used to compare the binding profiles have not yet been fully explored or optimized. As a result, binding profiles depend on TF structural information and sometimes may ignore important distinctions between subfamilies. Prediction of the identity or the structural class of a protein that binds to a given DNA pattern will enhance the analysis of microarray and ChIP-chip data where frequently multiple putative targets of usually unknown TFs are predicted. Various comparison metrics and alignment algorithms are evaluated (a total of 105 combinations). We find that local alignments are generally better than global alignments at detecting eukaryotic DNA motif similarities, especially when combined with the sum of squared distances or Pearson's correlation coefficient comparison metrics. In addition, multiple-alignment strategies for binding profiles and tree-building methods are tested for their efficiency in constructing generalized binding models. A new method for automatic determination of the optimal number of clusters is developed and applied in the construction of a new set of familial binding profiles which improves upon TF classification accuracy. A software tool, STAMP, is developed to host all tested methods and make them publicly available. This work provides a high quality reference set of familial binding profiles and the first comprehensive platform for analysis of DNA profiles. Detecting similarities between DNA motifs is a key step in the comparative study

  11. Analysis of protective antigen peptide binding motifs using bacterial display technology

    NASA Astrophysics Data System (ADS)

    Sarkes, Deborah A.; Dorsey, Brandi L.; Stratis-Cullum, Dimitra N.

    2015-05-01

    In today's fast-paced world, a new biological threat could emerge at any time, necessitating a prompt, reliable, inexpensive detection reagent in each case. Combined with magnetic-activated cell sorting (MACS), bacterial display technology makes it possible to isolate selective, high affinity peptide reagents in days to weeks. Utilizing the eCPX display scaffold is also a rapid way to screen potential peptide reagents. Peptide affinity reagents for protective antigen (PA) of the biothreat Bacillus anthracis were previously discovered using bacterial display. Bioinformatics analysis resulted in the consensus sequence WXCFTC. Additionally, we have discovered PA binding peptides with a WW motif, one of which, YGLHPWWKNAPIGQR, can pull down PA from 1% human serum. The strength of these two motifs combined, to obtain a WWCFTC consensus, is assessed here using Fluorescence Activated Cell Sorting (FACS). While monitoring binding to PA, overall expression of the display scaffold was assessed using the YPet Mona expression control tag (YPet), and specificity was assessed by binding to Streptavidin R-Phycoerythrin (SAPE). The importance of high YPet binding is highlighted as many of the peptides in one of the three replicate experiments fell below our 80% binding threshold. We demonstrate that it is preferable to discard this experiment, due to questionable expression of the peptide itself, than to try to normalize for relative expression. The peptides containing the WWCFTC consensus were of higher affinity and greater specificity than the peptides containing the WW consensus alone, validating further investigation to optimize known PA binders.

  12. Automatic generation of 3D motifs for classification of protein binding sites

    PubMed Central

    Nebel, Jean-Christophe; Herzyk, Pawel; Gilbert, David R

    2007-01-01

    Background Since many of the new protein structures delivered by high-throughput processes do not have any known function, there is a need for structure-based prediction of protein function. Protein 3D structures can be clustered according to their fold or secondary structures to produce classes of some functional significance. A recent alternative has been to detect specific 3D motifs which are often associated to active sites. Unfortunately, there are very few known 3D motifs, which are usually the result of a manual process, compared to the number of sequential motifs already known. In this paper, we report a method to automatically generate 3D motifs of protein structure binding sites based on consensus atom positions and evaluate it on a set of adenine based ligands. Results Our new approach was validated by generating automatically 3D patterns for the main adenine based ligands, i.e. AMP, ADP and ATP. Out of the 18 detected patterns, only one, the ADP4 pattern, is not associated with well defined structural patterns. Moreover, most of the patterns could be classified as binding site 3D motifs. Literature research revealed that the ADP4 pattern actually corresponds to structural features which show complex evolutionary links between ligases and transferases. Therefore, all of the generated patterns prove to be meaningful. Each pattern was used to query all PDB proteins which bind either purine based or guanine based ligands, in order to evaluate the classification and annotation properties of the pattern. Overall, our 3D patterns matched 31% of proteins with adenine based ligands and 95.5% of them were classified correctly. Conclusion A new metric has been introduced allowing the classification of proteins according to the similarity of atomic environment of binding sites, and a methodology has been developed to automatically produce 3D patterns from that classification. A study of proteins binding adenine based ligands showed that these 3D patterns are not

  13. Targeting a DNA binding motif of the EVI1 protein by a pyrrole-imidazole polyamide.

    PubMed

    Zhang, Yi; Sicot, Géraldine; Cui, Xiaohui; Vogel, Marion; Wuertzer, Charles A; Lezon-Geyda, Kimberly; Wheeler, John; Harki, Daniel A; Muzikar, Katy A; Stolper, Daniel A; Dervan, Peter B; Perkins, Archibald S

    2011-12-06

    The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole-imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure-function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1-7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole-imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.

  14. Nucleotide binding database NBDB – a collection of sequence motifs with specific protein-ligand interactions

    PubMed Central

    Zheng, Zejun; Goncearenco, Alexander; Berezovsky, Igor N.

    2016-01-01

    NBDB database describes protein motifs, elementary functional loops (EFLs) that are involved in binding of nucleotide-containing ligands and other biologically relevant cofactors/coenzymes, including ATP, AMP, ATP, GMP, GDP, GTP, CTP, PAP, PPS, FMN, FAD(H), NAD(H), NADP, cAMP, cGMP, c-di-AMP and c-di-GMP, ThPP, THD, F-420, ACO, CoA, PLP and SAM. The database is freely available online at http://nbdb.bii.a-star.edu.sg. In total, NBDB contains data on 249 motifs that work in interactions with 24 ligands. Sequence profiles of EFL motifs were derived de novo from nonredundant Uniprot proteome sequences. Conserved amino acid residues in the profiles interact specifically with distinct chemical parts of nucleotide-containing ligands, such as nitrogenous bases, phosphate groups, ribose, nicotinamide, and flavin moieties. Each EFL profile in the database is characterized by a pattern of corresponding ligand–protein interactions found in crystallized ligand–protein complexes. NBDB database helps to explore the determinants of nucleotide and cofactor binding in different protein folds and families. NBDB can also detect fragments that match to profiles of particular EFLs in the protein sequence provided by user. Comprehensive information on sequence, structures, and interactions of EFLs with ligands provides a foundation for experimental and computational efforts on design of required protein functions. PMID:26507856

  15. Relevant role of fibronectin-binding proteins in Staphylococcus aureus biofilm-associated foreign-body infections.

    PubMed

    Vergara-Irigaray, Marta; Valle, Jaione; Merino, Nekane; Latasa, Cristina; García, Begoña; Ruiz de Los Mozos, Igor; Solano, Cristina; Toledo-Arana, Alejandro; Penadés, José R; Lasa, Iñigo

    2009-09-01

    Staphylococcus aureus can establish chronic infections on implanted medical devices due to its capacity to form biofilms. Analysis of the factors that assemble cells into a biofilm has revealed the occurrence of strains that produce either a polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide- or a protein-dependent biofilm. Examination of the influence of matrix nature on the biofilm capacities of embedded bacteria has remained elusive, because a natural strain that readily converts between a polysaccharide- and a protein-based biofilm has not been studied. Here, we have investigated the clinical methicillin (meticillin)-resistant Staphylococcus aureus strain 132, which is able to alternate between a proteinaceous and an exopolysaccharidic biofilm matrix, depending on environmental conditions. Systematic disruption of each member of the LPXTG surface protein family identified fibronectin-binding proteins (FnBPs) as components of a proteinaceous biofilm formed in Trypticase soy broth-glucose, whereas a PIA/PNAG-dependent biofilm was produced under osmotic stress conditions. The induction of FnBP levels due to a spontaneous agr deficiency present in strain 132 and the activation of a LexA-dependent SOS response or FnBP overexpression from a multicopy plasmid enhanced biofilm development, suggesting a direct relationship between the FnBP levels and the strength of the multicellular phenotype. Scanning electron microscopy revealed that cells growing in the FnBP-mediated biofilm formed highly dense aggregates without any detectable extracellular matrix, whereas cells in a PIA/PNAG-dependent biofilm were embedded in an abundant extracellular material. Finally, studies of the contribution of each type of biofilm matrix to subcutaneous catheter colonization revealed that an FnBP mutant displayed a significantly lower capacity to develop biofilm on implanted catheters than the isogenic PIA/PNAG-deficient mutant.

  16. Production of Fibronectin Binding Protein A at the surface of Lactococcus lactis increases plasmid transfer in vitro and in vivo.

    PubMed

    Pontes, Daniela; Innocentin, Silvia; Del Carmen, Silvina; Almeida, Juliana Franco; Leblanc, Jean-Guy; de Moreno de Leblanc, Alejandra; Blugeon, Sébastien; Cherbuy, Claire; Lefèvre, François; Azevedo, Vasco; Miyoshi, Anderson; Langella, Philippe; Chatel, Jean-Marc

    2012-01-01

    Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cow's milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.

  17. Dynamic Structure of Plasma Fibronectin

    PubMed Central

    Maurer, Lisa M.; Ma, Wenjiang; Mosher, Deane F.

    2016-01-01

    Fibronectin is a large vertebrate glycoprotein that is found in soluble and insoluble forms and involved in diverse processes. Protomeric fibronectin is a dimer of subunits, each of which comprises 29 to 31 modules—12 type I, two type II, and 15-17 type III. Plasma fibronectin is secreted by hepatocytes and circulates in a compact conformation before it binds to cell surfaces, converts to an extended conformation, and is assembled into fibronectin fibrils. Here we review biophysical and structural studies that have shed light on how plasma fibronectin transitions from the compact to the extended conformation. The three types of modules each have a well-organized secondary and tertiary structure as defined by NMR and crystallography and have been likened to “beads on a string”. There are flexible sequences in the N-terminal tail, between the fifth and sixth type I modules, between the first two and last two of the type III modules, and at the C-terminus. Several specific module-module interactions have been identified that likely maintain the compact quaternary structure of circulating fibronectin. The quaternary structure is perturbed in response to binding events, including binding of fibronectin to the surface of vertebrate cells for fibril assembly and to bacterial adhesins. PMID:27185500

  18. Identification of an Orthogonal Peptide Binding Motif for Biarsenical Multiuse Affinity Probes

    SciTech Connect

    Chen, Baowei; Cao, Haishi; Yan, Ping; Mayer, M. Uljana; Squier, Thomas C.

    2007-07-01

    Biarsenical multiuse affinity probes (MAPs) complexed with ethanedithiol (EDT) permit the selective cellular labeling of proteins engineered with tetracysteine motifs, but are limited by the availability of a single binding motif (i.e., CCPGCC or PG tag) that prevents the differential labeling of co-expressed proteins. To overcome this problem, we have used a high-throughput peptide screen to identify an alternate binding motif (i.e., CCKACC or KA tag), which has a similar brightness to the classical sequence upon MAP binding, but displays altered rates and affinities of association that permit the differential labeling of these peptide sequences by the red probe 4,5-bis(1,3,2-dithiarsolan-2-yl)-resorufin (ReAsH-EDT2) or its green cognate 4’,5’-bis(1,3,2-dithoarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2 (FLAsH-EDT2). The utility of this labeling strategy was demonstrated following the expression of PG- and KA-tagged subunits of RNA polymerase expressed in E. coli. Specific labeling of two subunits of RNA polymerase in cellular lysates was achieved, whereby ReAsH-EDT2 is shown to selectively label the PG-tag on RNA polymerase alpha subunit prior to the labeling of the KA-tag sequence of the beta subunit of RNA polymerase with FlAsH-EDT2. These results demonstrate the ability to selectively label multiple individual proteins with orthogonal sequence tags in complex cellular lystates with spectroscopically distinct MAPs, and indicate the absolute specificity of ReAsH to target expressed proteins with essentially no nonspecific binding interactions.

  19. GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

    PubMed

    He, Ximiao; Syed, Khund Sayeed; Tillo, Desiree; Mann, Ishminder; Weirauch, Matthew T; Vinson, Charles

    2015-07-16

    To achieve proper spatiotemporal control of gene expression, transcription factors cooperatively assemble onto specific DNA sequences. The ETS domain protein monomer of GABPα and the B-ZIP domain protein dimer of CREB1 cooperatively bind DNA only when the ETS ((C)/GCGGAA GT: ) and CRE ( GT: GACGTCAC) motifs overlap precisely, producing the ETS↔CRE motif ((C)/GCGGAA GT: GACGTCAC). We designed a Protein Binding Microarray (PBM) with 60-bp DNAs containing four identical sectors, each with 177,440 features that explore the cooperative interactions between GABPα and CREB1 upon binding the ETS↔CRE motif. The DNA sequences include all 15-mers of the form (C)/GCGGA--CG-, the ETS↔CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes. CREB1 enhanced GABPα binding to the canonical ETS↔CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding. We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABPα and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding.

  20. Local motifs involved in the canonical structure of the ligand-binding domain in the nuclear receptor superfamily.

    PubMed

    Tsuji, Motonori

    2014-03-01

    Structural and sequence alignment analyses have revealed the existence of class-dependent and -independent local motifs involved in the overall fold of the ligand-binding domain (LBD) in the nuclear receptor (NR) superfamily. Of these local motifs, three local motifs, i.e., AF-2 fixed motifs, were involved in the agonist conformation of the activation function-2 (AF-2) region of the LBD. Receptor-agonist interactions increased the stability of these AF-2 fixed motifs in the agonist conformation. In contrast, perturbation of the AF-2 fixed motifs by a ligand or another protein molecule led the AF-2 architecture to adopt an antagonist conformation. Knowledge of this process should provide us with novel insights into the 'agonism' and 'antagonism' of NRs.

  1. Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen.

    PubMed

    Konitsiotis, Antonios D; Raynal, Nicolas; Bihan, Dominique; Hohenester, Erhard; Farndale, Richard W; Leitinger, Birgit

    2008-03-14

    The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.

  2. Trans-regulation of RNA-binding protein motifs by microRNA

    PubMed Central

    Doyle, Francis; Tenenbaum, Scott A.

    2014-01-01

    The wide array of vital functions that RNA performs is dependent on its ability to dynamically fold into different structures in response to intracellular and extracellular changes. RNA-binding proteins regulate much of this activity by targeting specific RNA structures or motifs. One of these structures, the 3-way RNA junction, is characteristically found in ribosomal RNA and results from the RNA folding in cis, to produce three separate helices that meet around a central unpaired region. Here we demonstrate that 3-way junctions can also form in trans as a result of the binding of microRNAs in an unconventional manner with mRNA by splinting two non-contiguous regions together. This may be used to reinforce the base of a stem-loop motif being targeted by an RNA-binding protein. Trans interactions between non-coding RNA and mRNA may be used to control the post-transcriptional regulatory code and suggests a possible role for some of the recently described transcripts of unknown function expressed from the human genome. PMID:24795744

  3. The 'helix clamp' in HIV-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases.

    PubMed Central

    Hermann, T; Meier, T; Götte, M; Heumann, H

    1994-01-01

    Amino acid sequences homologous to 259KLVGKL (X)16KLLR284 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) are conserved in several nucleotide polymerizing enzymes. This amino acid motif has been identified in the crystal structure model as an element of the enzyme's nucleic acid binding apparatus. It is part of the helix-turn-helix structure, alpha H-turn-alpha I, within the 'thumb' region of HIV-1 RT. The motif grasps the complexed nucleic acid at one side. Molecular modeling studies on HIV-1 RT in complex with a nucleic acid fragment suggest that the motif has binding function in the p66 subunit as well as in the p51 subunit, acting as a kind of 'helix clamp'. Given its wide distribution within the nucleic acid polymerases, the helix clamp motif is assumed to be a structure of general significance for nucleic acid binding. Images PMID:7527138

  4. Insight into centromere-binding properties of ParB proteins: a secondary binding motif is essential for bacterial genome maintenance

    PubMed Central

    Sanchez, Aurore; Rech, Jérôme; Gasc, Cyrielle; Bouet, Jean-Yves

    2013-01-01

    ParB proteins are one of the three essential components of partition systems that actively segregate bacterial chromosomes and plasmids. In binding to centromere sequences, ParB assembles as nucleoprotein structures called partition complexes. These assemblies are the substrates for the partitioning process that ensures DNA molecules are segregated to both sides of the cell. We recently identified the sopC centromere nucleotides required for binding to the ParB homologue of plasmid F, SopB. This analysis also suggested a role in sopC binding for an arginine residue, R219, located outside the helix-turn-helix (HTH) DNA-binding motif previously shown to be the only determinant for sopC-specific binding. Here, we demonstrated that the R219 residue is critical for SopB binding to sopC during partition. Mutating R219 to alanine or lysine abolished partition by preventing partition complex assembly. Thus, specificity of SopB binding relies on two distinct motifs, an HTH and an arginine residue, which define a split DNA-binding domain larger than previously thought. Bioinformatic analysis over a broad range of chromosomal ParBs generalized our findings with the identification of a non-HTH positively charged residue essential for partition and centromere binding, present in a newly identified highly conserved motif. We propose that ParB proteins possess two DNA-binding motifs that form an extended centromere-binding domain, providing high specificity. PMID:23345617

  5. UafB is a serine-rich repeat adhesin of Staphylococcus saprophyticus that mediates binding to fibronectin, fibrinogen and human uroepithelial cells.

    PubMed

    King, Nathan P; Beatson, Scott A; Totsika, Makrina; Ulett, Glen C; Alm, Richard A; Manning, Paul A; Schembri, Mark A

    2011-04-01

    Staphylococcus saprophyticus is an important cause of urinary tract infection (UTI), particularly among young women, and is second only to uropathogenic Escherichia coli as the most frequent cause of UTI. The molecular mechanisms of urinary tract colonization by S. saprophyticus remain poorly understood. We have identified a novel 6.84 kb plasmid-located adhesin-encoding gene in S. saprophyticus strain MS1146 which we have termed uro-adherence factor B (uafB). UafB is a glycosylated serine-rich repeat protein that is expressed on the surface of S. saprophyticus MS1146. UafB also functions as a major cell surface hydrophobicity factor. To characterize the role of UafB we generated an isogenic uafB mutant in S. saprophyticus MS1146 by interruption with a group II intron. The uafB mutant had a significantly reduced ability to bind to fibronectin and fibrinogen. Furthermore, we show that a recombinant protein containing the putative binding domain of UafB binds specifically to fibronectin and fibrinogen. UafB was not involved in adhesion in a mouse model of UTI; however, we observed a striking UafB-mediated adhesion phenotype to human uroepithelial cells. We have also identified genes homologous to uafB in other staphylococci which, like uafB, appear to be located on transposable elements. Thus, our data indicate that UafB is a novel adhesin of S. saprophyticus that contributes to cell surface hydrophobicity, mediates adhesion to fibronectin and fibrinogen, and exhibits tropism for human uroepithelial cells.

  6. Fibronectin of human liver sinusoids binds hepatitis B virus: identification by an anti-idiotypic antibody bearing the internal image of the pre-S2 domain.

    PubMed Central

    Budkowska, A; Bedossa, P; Groh, F; Louise, A; Pillot, J

    1995-01-01

    Anti-idiotypic antibodies (anti-Ids) have been successfully used to characterize and isolate receptors of several cell ligands. To prepare an immunological probe for identification of cellular components interacting with the hepatitis B virus (HBV), polyclonal antisera against a panel of five HBV-specific monoclonal antibodies (MAbs) were produced in syngeneic BALB/c mice. MAbs to HBV used for immunization (Ab1) recognized biologically important and potentially neutralizing epitopes, located in the pre-S1, pre-S2, or S region-encoded domains of HBV proteins. All the anti-Ids (Ab2) were specific to idiotopes of the homologous Ab1 and inhibited their interaction with the corresponding viral epitopes, suggesting that they recognized unique determinants on the paratope of each immunizing Ab1. Therefore, all five generated polyclonal anti-Ids were of the Ab2 beta type and could represent internal images of viral epitopes. Ab2 raised against the pre-S2 region-specific MAb F124 bound to the extracellular matrix fibronectin of human liver sinusoids. Immunohistochemical studies demonstrated the attachment of viral and recombinant (S, M) hepatitis B surface antigen particles with the pre-S2 region-encoded epitopes to the fibronectin of human liver sinusoids. In contrast, recombinant (S, L*) hepatitis B surface antigen particles, in which the epitope recognized by F124 MAb was not expressed, did not show any binding capacity. These findings suggest that human liver fibronectin may bind HBV in vivo by the pre-S2 region-encoded epitopes in a species-restricted manner. Furthermore, binding of the circulating virus to liver sinusoids could facilitate its subsequent uptake by hepatocytes. PMID:7815551

  7. Multiple Binding Modes between HNF4α and the LXXLL Motifs of PGC-1α Lead to Full Activation*

    PubMed Central

    Rha, Geun Bae; Wu, Guangteng; Shoelson, Steven E.; Chi, Young-In

    2009-01-01

    Hepatocyte nuclear factor 4α (HNF4α) is a novel nuclear receptor that participates in a hierarchical network of transcription factors regulating the development and physiology of such vital organs as the liver, pancreas, and kidney. Among the various transcriptional coregulators with which HNF4α interacts, peroxisome proliferation-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) represents a novel coactivator whose activation is unusually robust and whose binding mode appears to be distinct from that of canonical coactivators such as NCoA/SRC/p160 family members. To elucidate the potentially unique molecular mechanism of PGC-1α recruitment, we have determined the crystal structure of HNF4α in complex with a fragment of PGC-1α containing all three of its LXXLL motifs. Despite the presence of all three LXXLL motifs available for interactions, only one is bound at the canonical binding site, with no additional contacts observed between the two proteins. However, a close inspection of the electron density map indicates that the bound LXXLL motif is not a selected one but an averaged structure of more than one LXXLL motif. Further biochemical and functional studies show that the individual LXXLL motifs can bind but drive only minimal transactivation. Only when more than one LXXLL motif is involved can significant transcriptional activity be measured, and full activation requires all three LXXLL motifs. These findings led us to propose a model wherein each LXXLL motif has an additive effect, and the multiple binding modes by HNF4α toward the LXXLL motifs of PGC-1α could account for the apparent robust activation by providing a flexible mechanism for combinatorial recruitment of additional coactivators and mediators. PMID:19846556

  8. Magnetite-Binding Flagellar Filaments Displaying the MamI Loop Motif.

    PubMed

    Bereczk-Tompa, Éva; Pósfai, Mihály; Tóth, Balázs; Vonderviszt, Ferenc

    2016-11-03

    This work aimed at developing a novel method for fabricating 1 D magnetite nanostructures with the help of mutated flagellar filaments. We constructed four different flagellin mutants displaying magnetite-binding motifs: two contained fragments of magnetosome-associated proteins from magnetotactic bacteria (MamI and Mms6), and synthetic sequences were used for the other two. A magnetic selection method identified the MamI mutant as having the highest binding affinity to magnetite. Filaments built from MamI loop-containing flagellin subunits were used as templates to form chains of magnetite nanoparticles along the filament by capturing them from suspension. Our study represents a proof-of-concept that flagellar filaments can be engineered to facilitate formation of 1 D magnetite nanostructures under ambient conditions. In addition, it proves the interaction between MamI and magnetite, with implications for the role of this protein in magnetotactic bacteria.

  9. Binding Mode of Acetylated Histones to Bromodomains: Variations on a Common Motif.

    PubMed

    Marchand, Jean-Rémy; Caflisch, Amedeo

    2015-08-01

    Bromodomains, epigenetic readers that recognize acetylated lysine residues in histone tails, are potential drug targets in cancer and inflammation. Herein we review the crystal structures of human bromodomains in complex with histone tails and analyze the main interaction motifs. The histone backbone is extended and occupies, in one of the two possible orientations, the bromodomain surface groove lined by the ZA and BC loops. The acetyl-lysine side chain is buried in the cavity between the four helices of the bromodomain, and its oxygen atom accepts hydrogen bonds from a structural water molecule and a conserved asparagine residue in the BC loop. In stark contrast to this common binding motif, a large variety of ancillary interactions emerge from our analysis. In 10 of 26 structures, a basic side chain (up to five residues up- or downstream in sequence with respect to the acetyl-lysine) interacts with the carbonyl groups of the C-terminal turn of helix αB. Furthermore, the complexes reveal many heterogeneous backbone hydrogen bonds (direct or water-bridged). These interactions contribute unselectively to the binding of acetylated histone tails to bromodomains, which provides further evidence that specific recognition is modulated by combinations of multiple histone modifications and multiple modules of the proteins involved in transcription.

  10. 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.

  11. Individual RNA recognition motifs of TIA-1 and TIAR have different RNA binding specificities.

    PubMed

    Dember, L M; Kim, N D; Liu, K Q; Anderson, P

    1996-02-02

    TIA-1 and TIAR are two closely related RNA recognition motif (RRM) proteins which possess three RRM-type RNA binding domains (RRMs 1, 2, and 3). Although both proteins have been implicated as effectors of apoptotic cell death, the specific functions of TIA-1 and TIAR are not known. We have performed in vitro selection/amplification from pools of random RNA sequences to identify RNAs to which TIA-1 and TIAR bind with high affinity. Both proteins selected RNAs containing one or several short stretches of uridylate residues suggesting that the two proteins have similar RNA binding specificities. Replacement of the uridylate stretch with an equal number of cytidine residues eliminates the protein-RNA interaction. Mutational analysis indicates that, for both TIA-1 and TIAR, it is the second RNA binding domain (RRM 2) which mediates the specific binding to uridylate-rich RNAs. Although RRM 2 is both necessary and sufficient for this interaction, the affinity for the selected RNA (as determined by filter binding assays) does increase when the second domain of TIAR is expressed together with the first and third domains (Kd = 2 x 10(-8) M) rather than alone (Kd = 5 x 10(-8) M). Although RRM 3 (of either TIA-1 or TIAR) does not interact with the uridylate-rich sequences selected by the full-length proteins, it is a bona fide RNA binding domain capable of affinity-precipitating a population of cellular RNAs ranging in size from 0.5 to 5 kilobases. In contrast, RRM 1 does not affinity-precipitate cellular RNA. The inability of RRM 1 to interact with RNA may be due to the presence of negatively charged amino acids within the RNP 1 octamer.

  12. Hydrogen Bonding Motifs in MutSaphla and their response to binding damaged DNA

    NASA Astrophysics Data System (ADS)

    Negureanu, Lacra; Salsbury, Freddie

    2013-03-01

    Over the past decade, there has been a growing interest in studying the binding of damaged DNA to the MutSalpha protein complex. This protein complex, the Msh2/Msh6 complex in humans, is the initial complex that binds mismatched DNA and other DNA defects that occur during replication. This complex has also been shown to bind at least some types of damaged DNA. As a result of this interest, multiple studies have contrasted the interactions of MutSalpha with its normal mismatched substrate and with the interactions of MutsSalpha to DNA damaged by the chemotherapeutic cisplatin. To complement these studies, we examined the interaction between MutSalpha and DNA damaged by carboplatin via all-atom molecular dynamics simulations. These simulations provide evidence for different hydrogen bonding interactions at the protein/DNA and protein/protein interface. The hydrogen bonding motifs found are broadly similar to those found in binding to the adduct from cis-platin, but have distinct differences. These subtle differences may play a role in the way the different damages are signaled by MutS.

  13. Reconstitution of the receptor-binding motif of the SARS coronavirus.

    PubMed

    Freund, Natalia T; Roitburd-Berman, Anna; Sui, Jianhua; Marasco, Wayne A; Gershoni, Jonathan M

    2015-12-01

    The severe acute respiratory syndrome (SARS) coronavirus (CoV) identified in 2003 has infected ∼8000 people worldwide, killing nearly 10% of them. The infection of target cells by the SARS CoV is mediated through the interaction of the viral Spike (S) protein (1255 amino acids) and its cellular receptor, angiotensin-converting enzyme 2 (ACE2). The SARS CoV receptor-binding domain (amino acids N318-T509 of S protein) harbors an extended excursion along its periphery that contacts ACE2 and is designated the receptor-binding motif (RBM, amino acids S432-T486). In addition, the RBM is a major antigenic determinant, able to elicit production of neutralizing antibodies. Hence, the role of the RBM is a bi-functional bioactive surface that can be demonstrated by antibodies such as the neutralizing human anti-SARS monoclonal antibody (mAb) 80R which targets the RBM and competes with the ACE2 receptor for binding. Here, we employ phage-display peptide-libraries to reconstitute a functional RBM. This is achieved by generating a vast collection of candidate RBM peptides that present a diversity of conformations. Screening such 'Conformer Libraries' with corresponding ligands has produced short RBM constructs (ca. 40 amino acids) that can bind both the ACE2 receptor and the neutralizing mAb 80R.

  14. A Siglec-like sialic-acid-binding motif revealed in an adenovirus capsid protein

    PubMed Central

    Rademacher, Christoph; Bru, Thierry; McBride, Ryan; Robison, Elizabeth; Nycholat, Corwin M; Kremer, Eric J; Paulson, James C

    2012-01-01

    Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acα2-3[6S]Galβ1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid. PMID:22522600

  15. Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding

    PubMed Central

    Boddey, Justin A.; O'Neill, Matthew T.; Lopaticki, Sash; Carvalho, Teresa G.; Hodder, Anthony N.; Nebl, Thomas; Wawra, Stephan; van West, Pieter; Ebrahimzadeh, Zeinab; Richard, Dave; Flemming, Sven; Spielmann, Tobias; Przyborski, Jude; Babon, Jeff J.; Cowman, Alan F.

    2016-01-01

    Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum. Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export. PMID:26832821

  16. Identification of HI-like loop in CELO adenovirus fiber for incorporation of receptor binding motifs.

    PubMed

    Logunov, Denis Y; Zubkova, Olga V; Karyagina-Zhulina, Anna S; Shuvalova, Eugenia A; Karpov, Andrei P; Shmarov, Maxim M; Tutykhina, Irina L; Alyapkina, Yulia S; Grezina, Natalia M; Zinovieva, Natalia A; Ernst, Lev K; Gintsburg, Alexsandr L; Naroditsky, Boris S

    2007-09-01

    Vectors based on the chicken embryo lethal orphan (CELO) avian adenovirus (Ad) have two attractive properties for gene transfer applications: resistance to preformed immune responses to human Ads and the ability to grow in chicken embryos, allowing low-cost production of recombinant viruses. However, a major limitation of this technology is that CELO vectors demonstrate decreased efficiency of gene transfer into cells expressing low levels of the coxsackie-Ad receptor (CAR). In order to improve the efficacy of gene transfer into CAR-deficient cells, we modified viral tropism via genetic alteration of the CELO fiber 1 protein. The alphav integrin-binding motif (RGD) was incorporated at two different sites of the fiber 1 knob domain, within an HI-like loop that we identified and at the C terminus. Recombinant fiber-modified CELO viruses were constructed containing secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein genes as reporter genes. Our data show that insertion of the RGD motif within the HI-like loop of the fiber resulted in significant enhancement of gene transfer into CAR-negative and CAR-deficient cells. In contrast, CELO vectors containing the RGD motif at the fiber 1 C terminus showed reduced transduction of all cell lines. CELO viruses modified with RGD at the HI-like loop transduced the SEAP reporter gene into rabbit mammary gland cells in vivo with an efficiency significantly greater than that of unmodified CELO vector and similar to that of Ad type 5 vector. These results illustrate the potential for efficient CELO-mediated gene transfer into a broad range of cell types through modification of the identified HI-like loop of the fiber 1 protein.

  17. Identification by Flagellum Display of an Epithelial Cell- and Fibronectin-Binding Function in the SlpA Surface Protein of Lactobacillus brevis

    PubMed Central

    Hynönen, Ulla; Westerlund-Wikström, Benita; Palva, Airi; Korhonen, Timo K.

    2002-01-01

    Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliCH7 gene of Escherichia coli. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of L. brevis ATCC 8287 as an adhesin with affinity

  18. Improved bioactivity of antimicrobial peptides by addition of amino-terminal copper and nickel (ATCUN) binding motifs.

    PubMed

    Libardo, M Daben; Cervantes, Jorge L; Salazar, Juan C; Angeles-Boza, Alfredo M

    2014-08-01

    Antimicrobial peptides (AMPs) are promising candidates to help circumvent antibiotic resistance, which is an increasing clinical problem. Amino-terminal copper and nickel (ATCUN) binding motifs are known to actively form reactive oxygen species (ROS) upon metal binding. The combination of these two peptidic constructs could lead to a novel class of dual-acting antimicrobial agents. To test this hypothesis, a set of ATCUN binding motifs were screened for their ability to induce ROS formation, and the most potent were then used to modify AMPs with different modes of action. ATCUN binding motif-containing derivatives of anoplin (GLLKRIKTLL-NH2), pro-apoptotic peptide (PAP; KLAKLAKKLAKLAK-NH2), and sh-buforin (RAGLQFPVGRVHRLLRK-NH2) were synthesized and found to be more active than the parent AMPs against a panel of clinically relevant bacteria. The lower minimum inhibitory concentration (MIC) values for the ATCUN-anoplin peptides are attributed to the higher pore-forming activity along with their ability to cause ROS-induced membrane damage. The addition of the ATCUN motifs to PAP also increases its ability to disrupt membranes. DNA damage is the major contributor to the activity of the ATCUN-sh-buforin peptides. Our findings indicate that the addition of ATCUN motifs to AMPs is a simple strategy that leads to AMPs with higher antibacterial activity and possibly to more potent, usable antibacterial agents.

  19. Associating transcription factor-binding site motifs with target GO terms and target genes

    PubMed Central

    Bodén, Mikael; Bailey, Timothy L.

    2008-01-01

    The roles and target genes of many transcription factors (TFs) are still unknown. To predict the roles of TFs, we present a computational method for associating Gene Ontology (GO) terms with TF-binding motifs. The method works by ranking all genes as potential targets of the TF, and reporting GO terms that are significantly associated with highly ranked genes. We also present an approach, whereby these predicted GO terms can be used to improve predictions of TF target genes. This uses a novel gene-scoring function that reflects the insight that genes annotated with GO terms predicted to be associated with the TF are more likely to be its targets. We construct validation sets of GO terms highly associated with known targets of various yeast and human TF. On the yeast reference sets, our prediction method identifies at least one correct GO term for 73% of the TF, 49% of the correct GO terms are predicted and almost one-third of the predicted GO terms are correct. Results on human reference sets are similarly encouraging. Validation of our target gene prediction method shows that its accuracy exceeds that of simple motif scanning. PMID:18544606

  20. Arabidopsis Polycomb Repressive Complex 2 binding sites contain putative GAGA factor binding motifs within coding regions of genes

    PubMed Central

    2013-01-01

    Background Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes. Results We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs). Conclusions Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3. PMID:24001316

  1. The Motif Tool Assessment Platform (MTAP) for sequence-based transcription factor binding site prediction tools.

    PubMed

    Quest, Daniel; Ali, Hesham

    2010-01-01

    Predicting transcription factor binding sites (TFBS) from sequence is one of the most challenging problems in computational biology. The development of (semi-)automated computer-assisted prediction methods is needed to find TFBS over an entire genome, which is a first step in reconstructing mechanisms that control gene activity. Bioinformatics journals continue to publish diverse methods for predicting TFBS on a monthly basis. To help practitioners in deciding which method to use to predict for a particular TFBS, we provide a platform to assess the quality and applicability of the available methods. Assessment tools allow researchers to determine how methods can be expected to perform on specific organisms or on specific transcription factor families. This chapter introduces the TFBS detection problem and reviews current strategies for evaluating algorithm effectiveness. In this chapter, a novel and robust assessment tool, the Motif Tool Assessment Platform (MTAP), is introduced and discussed.

  2. In vitro evolution of a peptide with a hematite binding motif that may constitute a natural metal-oxide binding archetype.

    PubMed

    Lower, Brian H; Lins, Roberto D; Oestreicher, Zachery; Straatsma, Tjerk P; Hochella, Michael F; Shi, Liang; Lower, Steven K

    2008-05-15

    Phage-display technology was used to evolve peptides that selectively bind to the metal-oxide hematite (Fe2O3) from a library of approximately 3 billion different polypeptides. The sequences of these peptides contained the highly conserved amino acid motif, Ser/Thr-hydrophobic/aromatic-Ser/Thr-Pro-Ser/Thr. To better understand the nature of the peptide-metal oxide binding demonstrated by these experiments, molecular dynamics simulations were carried out for Ser-Pro-Ser at a hematite surface. These simulations show that hydrogen bonding occurs between the two serine amino acids and the hydroxylated hematite surface and that the presence of proline between the hydroxide residues restricts the peptide flexibility, thereby inducing a structural-binding motif. A search of published sequence data revealed that the binding motif (Ser/Thr-Pro-Ser/Thr) is adjacent to the terminal heme-binding domain of both OmcA and MtrC, which are outer membrane cytochromes from the metal-reducing bacterium Shewanella oneidensis MR-1. The entire five amino acid consensus sequence (Ser/Thr-hydrophobic/ aromatic-Ser/Thr-Pro-Ser/Thr) was also found as multiple copies in the primary sequences of metal-oxide binding proteins Sil1 and Sil2 from Thalassiosira pseudonana. We suggest that this motif constitutes a natural metal-oxide binding archetype that could be exploited in enzyme-based biofuel cell design and approaches to synthesize tailored metal-oxide nanostructures.

  3. The structure of FKBP38 in complex with the MEEVD tetratricopeptide binding-motif of Hsp90

    PubMed Central

    Blundell, Katie L. I. M.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H.

    2017-01-01

    Tetratricopeptide (TPR) domains are known protein interaction domains. We show that the TPR domain of FKBP8 selectively binds Hsp90, and interactions upstream of the conserved MEEVD motif are critical for tight binding. In contrast FKBP8 failed to bind intact Hsp70. The PPIase domain was not essential for the interaction with Hsp90 and binding was completely encompassed by the TPR domain alone. The conformation adopted by Hsp90 peptides, containing the conserved MEEVD motif, in the crystal structure were similar to that seen for the TPR domains of CHIP, AIP and Tah1. The carboxylate clamp interactions with bound Hsp90 peptide were a critical component of the interaction and mutation of Lys 307, involved in the carboxylate clamp, completely disrupted the interaction with Hsp90. FKBP8 binding to Hsp90 did not substantially influence its ATPase activity. PMID:28278223

  4. A combined sequence and structure based method for discovering enriched motifs in RNA from in vivo binding data.

    PubMed

    Polishchuk, Maya; Paz, Inbal; Kohen, Refael; Mesika, Rona; Yakhini, Zohar; Mandel-Gutfreund, Yael

    2017-03-06

    RNA binding proteins (RBPs) play an important role in regulating many processes in the cell. RBPs often recognize their RNA targets in a specific manner. In addition to the RNA primary sequence, the structure of the RNA has been shown to play a central role in RNA recognition by RBPs. In recent years, many experimental approaches, both in vitro and in vivo, were developed and employed to identify and characterize RBP targets and extract their binding specificities. In vivo binding techniques, such as CrossLinking and ImmunoPrecipitation (CLIP)-based methods, enable the characterization of protein binding sites on RNA targets. However, these methods do not provide information regarding the structural preferences of the protein. While methods to obtain the structure of RNA are available, inferring both the sequence and the structure preferences of RBPs remains a challenge. Here we present SMARTIV, a novel computational tool for discovering combined sequence and structure binding motifs from in vivo RNA binding data relying on the sequences of the target sites, the ranking of their binding scores and their predicted secondary structure. The combined motifs are provided in a unified representation that is informative and easy for visual perception. We tested the method on CLIP-seq data from different platforms for a variety of RBPs. Overall, we show that our results are highly consistent with known binding motifs of RBPs, offering additional information on their structural preferences.

  5. The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

    SciTech Connect

    Zhang, Lei; Zhang, Qing; Yang, Yu; Wu, Chuanfang

    2014-02-14

    Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required for RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition.

  6. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators

    PubMed Central

    Hagting, Anja; Izawa, Daisuke; Mansfeld, Jörg; Gibson, Toby J.; Pines, Jonathon

    2016-01-01

    The APC/C is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the Spindle Assembly Checkpoint (SAC). How the APC/C recognises its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in Cyclin A, BUBR1, BUB1 and Acm1, and show that it binds to the APC/C co-activator CDC20. The ABBA motif in Cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC. PMID:25669885

  7. Enhanced Binding Affinity for an i-Motif DNA Substrate Exhibited by a Protein Containing Nucleobase Amino Acids.

    PubMed

    Bai, Xiaoguang; Talukder, Poulami; Daskalova, Sasha M; Roy, Basab; Chen, Shengxi; Li, Zhongxian; Dedkova, Larisa M; Hecht, Sidney M

    2017-03-17

    Several variants of a nucleic acid binding motif (RRM1) of putative transcription factor hnRNP LL containing nucleobase amino acids at specific positions have been prepared and used to study binding affinity for the BCL2 i-motif DNA. Molecular modeling suggested a number of amino acids in RRM1 likely to be involved in interaction with the i-motif DNA, and His24 and Arg26 were chosen for modification based on their potential ability to interact with G14 of the i-motif DNA. Four nucleobase amino acids were introduced into RRM1 at one or both of positions 24 and 26. The introduction of cytosine nucleobase 2 into position 24 of RRM1 increased the affinity of the modified protein for the i-motif DNA, consistent with the possible Watson-Crick interaction of 2 and G14. In comparison, the introduction of uracil nucleobase 3 had a minimal effect on DNA affinity. Two structurally simplified nucleobase analogues (1 and 4) lacking both the N-1 and the 2-oxo substituents were also introduced in lieu of His24. Again, the RRM1 analogue containing 1 exhibited enhanced affinity for the i-motif DNA, while the protein analogue containing 4 bound less tightly to the DNA substrate. Finally, the modified protein containing 1 in lieu of Arg26 also bound to the i-motif DNA more strongly than the wild-type protein, but a protein containing 1 both at positions 24 and 26 bound to the DNA less strongly than wild type. The results support the idea of using nucleobase amino acids as protein constituents for controlling and enhancing DNA-protein interaction. Finally, modification of the i-motif DNA at G14 diminished RRM1-DNA interaction, as well as the ability of nucleobase amino acid 1 to stabilize RRM1-DNA interaction.

  8. Elucidation of a C-rich signature motif in target mRNAs of RNA-binding protein TIAR.

    PubMed

    Kim, Henry S; Kuwano, Yuki; Zhan, Ming; Pullmann, Rudolf; Mazan-Mamczarz, Krystyna; Li, Huai; Kedersha, Nancy; Anderson, Paul; Wilce, Matthew C J; Gorospe, Myriam; Wilce, Jacqueline A

    2007-10-01

    The RNA-binding protein TIAR (related to TIA-1 [T-cell-restricted intracellular antigen 1]) was shown to associate with subsets of mRNAs bearing U-rich sequences in their 3' untranslated regions. TIAR can function as a translational repressor, particularly in response to cytotoxic agents. Using unstressed colon cancer cells, collections of mRNAs associated with TIAR were isolated by immunoprecipitation (IP) of (TIAR-RNA) ribonucleoprotein (RNP) complexes, identified by microarray analysis, and used to elucidate a common signature motif present among TIAR target transcripts. The predicted TIAR motif was an unexpectedly cytosine-rich, 28- to 32-nucleotide-long element forming a stem and a loop of variable size with an additional side loop. The ability of TIAR to bind an RNA oligonucleotide with a representative C-rich TIAR motif sequence was verified in vitro using surface plasmon resonance. By this analysis, TIAR containing two or three RNA recognition domains (TIAR12 and TIAR123) showed low but significant binding to the C-rich sequence. In vivo, insertion of the C-rich motif into a heterologous reporter strongly suppressed its translation in cultured cells. Using this signature motif, an additional approximately 2,209 UniGene targets were identified (2.0% of the total UniGene database). A subset of specific mRNAs were validated by RNP IP analysis. Interestingly, in response to treatment with short-wavelength UV light (UVC), a stress agent causing DNA damage, each of these target mRNAs bearing C-rich motifs dissociated from TIAR. In turn, expression of the encoded proteins was elevated in a TIAR-dependent manner. In sum, we report the identification of a C-rich signature motif present in TIAR target mRNAs whose association with TIAR decreases following exposure to a stress-causing agent.

  9. A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme?

    PubMed

    Koonin, E V; Bork, P; Sander, C

    1994-06-11

    Using computer methods for database search, multiple alignment, protein sequence motif analysis and secondary structure prediction, a putative new RNA-binding motif was identified. The novel motif is conserved in yeast omnipotent translation termination suppressor SUP1, the related DOM34 protein and its pseudogene homologue; three groups of eukaryotic and archaeal ribosomal proteins, namely L30e, L7Ae/S6e and S12e; an uncharacterized Bacillus subtilis protein related to the L7A/S6e group; and Escherichia coli ribosomal protein modification enzyme RimK. We hypothesize that a new type of RNA-binding domain may be utilized to deliver additional activities to the ribosome.

  10. Electrophoretic characterization of species of fibronectin bearing sequences from the N-terminal heparin-binding domain in synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis

    PubMed Central

    Peters, John H; Carsons, Steven; Yoshida, Mika; Ko, Fred; McDougall, Skye; Loredo, Grace A; Hahn, Theodore J

    2003-01-01

    Fragments of fibronectin (FN) corresponding to the N-terminal heparin-binding domain have been observed to promote catabolic chondrocytic gene expression and chondrolysis. We therefore characterized FN species that include sequences from this domain in samples of arthritic synovial fluid using one-and two-dimensional (1D and 2D) Western blot analysis. We detected similar assortments of species, ranging from ~47 to greater than 200 kDa, in samples obtained from patients with osteoarthritis (n = 9) versus rheumatoid arthritis (n = 10). One of the predominant forms, with an apparent molecular weight of ~170 kDa, typically resolved in 2D electrophoresis into a cluster of subspecies. These exhibited reduced binding to gelatin in comparison with a more prevalent species of ~200+ kDa and were also recognized by a monoclonal antibody to the central cell-binding domain (CBD). When considered together with our previous analyses of synovial fluid FN species containing the alternatively spliced EIIIA segment, these observations indicate that the ~170-kDa species includes sequences from four FN domains that have previously, in isolation, been observed to promote catabolic responses by chondrocytes in vitro: the N-terminal heparin-binding domain, the gelatin-binding domain, the central CBD, and the EIIIA segment. The ~170-kDa N-terminal species of FN may therefore be both a participant in joint destructive processes and a biomarker with which to gauge activity of the arthritic process. PMID:14680507

  11. The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif.

    PubMed

    Huth, J R; Bewley, C A; Nissen, M S; Evans, J N; Reeves, R; Gronenborn, A M; Clore, G M

    1997-08-01

    The solution structure of a complex between a truncated form of HMG-I(Y), consisting of the second and third DNA binding domains (residues 51-90), and a DNA dodecamer containing the PRDII site of the interferon-beta promoter has been solved by multidimensional nuclear magnetic resonance spectroscopy. The stoichiometry of the complex is one molecule of HMG-I(Y) to two molecules of DNA. The structure reveals a new architectural minor groove binding motif which stabilizes B-DNA, thereby facilitating the binding of other transcription factors in the opposing major groove. The interactions involve a central Arg-Gly-Arg motif together with two other modules that participate in extensive hydrophobic and polar contracts. The absence of one of these modules in the third DNA binding domain accounts for its-100 fold reduced affinity relative to the second one.

  12. Stb3 binds to ribosomal RNA processing element motifs that control transcriptional responses to growth in Saccharomyces cerevisiae.

    PubMed

    Liko, Dritan; Slattery, Matthew G; Heideman, Warren

    2007-09-07

    Transfer of quiescent Saccharomyces cerevisiae cells to fresh medium rapidly induces hundreds of genes needed for growth. A large subset of these genes is regulated via a DNA sequence motif known as the ribosomal RNA processing element (RRPE). However, no RRPE-binding proteins have been identified. We screened a panel of 6144 glutathione S-transferase-open reading frame fusions for RRPE-binding proteins and identified Stb3 as a specific RRPE-binding protein, both in vitro and in vivo. Chromatin immunoprecipitation experiments showed that glucose increases Stb3 binding to RRPE-containing promoters. Microarray experiments demonstrated that the loss of Stb3 inhibits the transcriptional response to fresh glucose, especially for genes with RRPE motifs. However, these experiments also showed that not all genes containing RRPEs were dependent on Stb3 for expression. Overall our data support a model in which Stb3 plays an important but not exclusive role in the transcriptional response to growth conditions.

  13. Binding of the C-terminal sterile alpha motif (SAM) domain of human p73 to lipid membranes.

    PubMed

    Barrera, Francisco N; Poveda, José A; González-Ros, José M; Neira, José L

    2003-11-21

    The alpha splice variant of p73 (p73alpha), a homologue of the tumor suppressor p53, has close to its C terminus a sterile alpha motif (SAM), SAMp73, that is thought to be involved in protein-protein interactions. Here, we report the lipid binding properties of this domain. Binding was assayed against zwitterionic (phosphatidylcholine) and anionic (phosphatidic acid) lipids and was studied by different biophysical techniques, namely, circular dichroism and fluorescence spectroscopies and differential scanning calorimetry. These techniques unambiguously indicate that SAMp73 binds to lipids. The binding involves protein surface attachment and partial membrane penetration, accompanied by changes in SAMp73 structure.

  14. The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

    PubMed Central

    Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

    2013-01-01

    AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences. PMID:23709277

  15. Cloning of a cellular factor, interleukin binding factor, that binds to NFAT-like motifs in the human immunodeficiency virus long terminal repeat.

    PubMed Central

    Li, C; Lai, C F; Sigman, D S; Gaynor, R B

    1991-01-01

    Human immunodeficiency virus (HIV) gene expression is regulated by both general transcription factors and factors induced by activation of T lymphocytes such as NF-kappa B and the nuclear factor of activated T cells (NFAT). Within the HIV long terminal repeat (LTR), two purine-rich domains between nucleotides -283 and -195 have homology to a regulatory region found in the interleukin 2 promoter, which binds NFAT and other cellular factors. In the HIV LTR, this region has been demonstrated to have both positive and negative regulatory effects on HIV gene expression. In an attempt to clone genes encoding cellular factors that bind to these NFAT-like elements in the HIV LTR, we used lambda gt11 expression cloning with oligonucleotides corresponding to these binding motifs. A ubiquitously expressed cDNA encoding a 60-kDa protein, which we termed interleukin binding factor (ILF), binds specifically to these purine-rich motifs in the HIV LTR. This factor also binds to similar purine-rich motifs in the interleukin 2 promoter, through with lower affinity than to HIV LTR sequences. Sequence analysis reveals that the DNA binding domain of ILF has strong homology to the recently described fork head DNA binding domain found in the Drosophila homeotic protein fork head and a family of hepatocyte nuclear factors, HNF-3. Other domains found in ILF include a nucleotide binding site, an N-glycosylation motif, a signal for ubiquitin-mediated degradation, and a potential nuclear localization signal. These results describe a DNA binding protein that may be involved in both positive and negative regulation of important viral and cellular promoter elements. Images PMID:1909027

  16. A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei

    PubMed Central

    1992-01-01

    The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule- associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins. PMID:1348252

  17. A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei.

    PubMed

    Hemphill, A; Affolter, M; Seebeck, T

    1992-04-01

    The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule-associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins.

  18. Binding motifs in bacterial gene promoters modulate transcriptional effect of global regulators

    SciTech Connect

    Leuze, Michael Rex; Karpinets, Tatiana V; Syed, Mustafa H; Beliaev, Alexander S; Uberbacher, Edward C

    2012-01-01

    Bacterial gene regulation involves transcription factors (TFs) that influence the expression of many genes. Global regulators, including CRP (cAMP Receptor Protein), ArcA, and FNR, can modulate the transcriptional activity of multiple operons. The similarity of a regulatory element s sequence to a TF s consensus binding site (BS) and the position of the regulatory element in an operon promoter are considered the most important determinants of this TF s regulatory influence. In this study we explore the hypothesis that the number of TFBS half-sites (where a half-site is one half of the palindromic BS consensus sequence, which we shall refer to as a binding motif or a BM) of a global regulator in an operon s promoter plays an important role in the operon s transcriptional regulation. We examine empirical data from transcriptional profiling of the CRP regulon in Shewanella oneidenses MR 1 and Escherichia coli, and of the ArcA regulon in S. oneidenses MR 1. We compare the power of CRP BM counts and of full, symmetrical CRP TFBS characteristics, namely similarity to consensus and location, to predict CRP-induced transcriptional activity. We find that CRP BM counts have a nonlinear effect on CRP-dependent transcriptional activity and predict this activity better than full-length TFBS quality or location. Regression analysis indicates that IHF (Integration Host Factor) and ArcA have synergistic effects on CRP-induced gene transcription, positive and negative, respectively. Based on these results, we propose that the fine-tuning of bacterial transcriptional activity by CRP may involves not only the bending of the operon promoter, facilitated by CRP in cooperation with the histone-like protein IHF, but also the cumulative binding affinity of multiple weak BMs.

  19. Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs.

    PubMed

    Kazakov, Alexei S; Sokolov, Andrei S; Vologzhannikova, Alisa A; Permyakova, Maria E; Khorn, Polina A; Ismailov, Ramis G; Denessiouk, Konstantin A; Denesyuk, Alexander I; Rastrygina, Victoria A; Baksheeva, Viktoriia E; Zernii, Evgeni Yu; Zinchenko, Dmitry V; Glazatov, Vladimir V; Uversky, Vladimir N; Mirzabekov, Tajib A; Permyakov, Eugene A; Permyakov, Sergei E

    2017-01-01

    Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1-19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, 'black' and 'gray' clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

  20. C-type lectin-like domain and fibronectin-like type II domain of phospholipase A(2) receptor 1 modulate binding and migratory responses to collagen.

    PubMed

    Takahashi, Soichiro; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-ei; Kugiyama, Kiyotaka

    2015-03-24

    Phospholipase A2 receptor 1 (PLA2R) mediates collagen-dependent migration. The mechanisms by which PLA2R interacts with collagen remain unclear. We produced HEK293 cells expressing full-length wild-type PLA2R or a truncated PLA2R that lacks fibronectin-like type II (FNII) domains or several regions of C-type lectin-like domain (CTLD). We show that the CTLD1-2 as well as the FNII domain of PLA2R are responsible for binding to collagen and for collagen-dependent migration. Thus, multiple regions and domains of the extracellular portion of PLA2R participate in the responses to collagen. These data suggest a potentially new mechanism for PLA2R-mediated biological response beyond that of a receptor for secretory PLA2.

  1. A fibronectin-binding protein (FbpA) of Weissella cibaria inhibits colonization and infection of Staphylococcus aureus in mammary glands.

    PubMed

    Wang, Liangliang; Si, Wei; Xue, Huping; Zhao, Xin

    2017-01-26

    Staphylococcus aureus (S. aureus) is a frequent cause of infections in both humans and animals. Probiotics are known to inhibit colonization of pathogens on host tissues. However, mechanisms for the inhibition are still elusive due to complex host-microbe and microbe-microbe interactions. Here, we show that reduced abilities of S. aureus to infect mammary glands in the presence of Weissella cibaria (W. cibaria) were correlated with its poor adherence to mammary epithelial cells. Such inhibition by W. cibaria isolates was at least partially attributed to a fibronectin-binding protein (FbpA) on this lactic acid bacterium. Three W. cibaria isolates containing fbpA had higher inhibitory abilities than other three LAB isolates without the gene. The fbpA-deficient mutant of W. cibaria isolate LW1, LW1ΔfbpA, lost the inhibitory activity to reduce the adhesion of S. aureus to mammary epithelial cells and was less able to reduce the colonization of S. aureus in mammary glands. Expression of FbpA to the surface of LW1ΔfbpA reversed its inhibitory activities. Furthermore, addition of purified FbpA inhibited S. aureus biofilm formation. Our results suggest that W. cibaria FbpA hinders S. aureus colonization and infection through interfering with the S. aureus invasion pathway mediated by fibronectin-binding proteins and inhibiting biofilm formation of S. aureus.

  2. DNA consensus sequence motif for binding response regulator PhoP, a virulence regulator of Mycobacterium tuberculosis.

    PubMed

    He, Xiaoyuan; Wang, Shuishu

    2014-12-30

    Tuberculosis has reemerged as a serious threat to human health because of the increasing prevalence of drug-resistant strains and synergetic infection with HIV, prompting an urgent need for new and more efficient treatments. The PhoP-PhoR two-component system of Mycobacterium tuberculosis plays an important role in the virulence of the pathogen and thus represents a potential drug target. To study the mechanism of gene transcription regulation by response regulator PhoP, we identified a high-affinity DNA sequence for PhoP binding using systematic evolution of ligands by exponential enrichment. The sequence contains a direct repeat of two 7 bp motifs separated by a 4 bp spacer, TCACAGC(N4)TCACAGC. The specificity of the direct-repeat sequence for PhoP binding was confirmed by isothermal titration calorimetry and electrophoretic mobility shift assays. PhoP binds to the direct repeat as a dimer in a highly cooperative manner. We found many genes previously identified to be regulated by PhoP that contain the direct-repeat motif in their promoter sequences. Synthetic DNA fragments at the putative promoter-binding sites bind PhoP with variable affinity, which is related to the number of mismatches in the 7 bp motifs, the positions of the mismatches, and the spacer and flanking sequences. Phosphorylation of PhoP increases the affinity but does not change the specificity of DNA binding. Overall, our results confirm the direct-repeat sequence as the consensus motif for PhoP binding and thus pave the way for identification of PhoP directly regulated genes in different mycobacterial genomes.

  3. The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain

    SciTech Connect

    Lechner, Mark S. . E-mail: msl27@drexel.edu; Schultz, David C.; Negorev, Dmitri; Maul, Gerd G.; Rauscher, Frank J.

    2005-06-17

    The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization.

  4. DNase I-hypersensitive sites and transcription factor-binding motifs within the mouse E beta meiotic recombination hot spot.

    PubMed

    Shenkar, R; Shen, M H; Arnheim, N

    1991-04-01

    The second intron of the E beta gene in the mouse major histocompatibility complex is the site of a meiotic recombination hot spot. We detected two DNase I-hypersensitive sites in this intron in meiotic cells isolated from mouse testes. One site appears to be constitutive and is found in other tissues regardless of whether or not they express the E beta gene. Near this hypersensitive site are potential binding motifs for H2TF1/KBF1, NF kappa B, and octamer transcription factors. Gel retardation studies with mouse lymphoma cell nuclear extracts confirmed that each of these motifs is capable of binding protein. The binding of transcription factors may contribute to the enhancement of recombination potential by altering chromatin structure and increasing the accessibility of the DNA to the recombination machinery.

  5. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

    SciTech Connect

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun Nishina, Hiroshi

    2014-01-17

    Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription.

  6. A signature motif in LIM proteins mediates binding to checkpoint proteins and increases tumour radiosensitivity

    PubMed Central

    Xu, Xiaojie; Fan, Zhongyi; Liang, Chaoyang; Li, Ling; Wang, Lili; Liang, Yingchun; Wu, Jun; Chang, Shaohong; Yan, Zhifeng; Lv, Zhaohui; Fu, Jing; Liu, Yang; Jin, Shuai; Wang, Tao; Hong, Tian; Dong, Yishan; Ding, Lihua; Cheng, Long; Liu, Rui; Fu, Shenbo; Jiao, Shunchang; Ye, Qinong

    2017-01-01

    Tumour radiotherapy resistance involves the cell cycle pathway. CDC25 phosphatases are key cell cycle regulators. However, how CDC25 activity is precisely controlled remains largely unknown. Here, we show that LIM domain-containing proteins, such as FHL1, increase inhibitory CDC25 phosphorylation by forming a complex with CHK2 and CDC25, and sequester CDC25 in the cytoplasm by forming another complex with 14-3-3 and CDC25, resulting in increased radioresistance in cancer cells. FHL1 expression, induced by ionizing irradiation in a SP1- and MLL1-dependent manner, positively correlates with radioresistance in cancer patients. We identify a cell-penetrating 11 amino-acid motif within LIM domains (eLIM) that is sufficient for binding CHK2 and CDC25, reducing the CHK2–CDC25 and CDC25–14-3-3 interaction and enhancing CDC25 activity and cancer radiosensitivity accompanied by mitotic catastrophe and apoptosis. Our results provide novel insight into molecular mechanisms underlying CDC25 activity regulation. LIM protein inhibition or use of eLIM may be new strategies for improving tumour radiosensitivity. PMID:28094252

  7. Membrane targeting of TIRAP is negatively regulated by phosphorylation in its phosphoinositide-binding motif

    PubMed Central

    Zhao, Xiaolin; Xiong, Wen; Xiao, Shuyan; Tang, Tuo-Xian; Ellena, Jeffrey F.; Armstrong, Geoffrey S.; Finkielstein, Carla V.; Capelluto, Daniel G. S.

    2017-01-01

    Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses. PMID:28225045

  8. A new calmodulin-binding motif for inositol 1,4,5-trisphosphate 3-kinase regulation.

    PubMed

    Franco-Echevarría, Elsa; Baños-Sanz, Jose I; Monterroso, Begoña; Round, Adam; Sanz-Aparicio, Julia; González, Beatriz

    2014-11-01

    IP3-3K [Ins(1,4,5)P3 3-kinase] is a key enzyme that catalyses the synthesis of Ins(1,3,4,5)P4, using Ins(1,4,5)P3 and ATP as substrates. Both inositides, substrate and product, present crucial roles in the cell. Ins(1,4,5)P3 is a key point in Ca2+ metabolism that promotes Ca2+ release from intracellular stores and together with Ins(1,3,4,5)P4 regulates Ca2+ homoeostasis. In addition, Ins(1,3,4,5)P4 is involved in immune cell development. It has been proved that Ca2+/CaM (calmodulin) regulates the activity of IP3-3K, via direct interaction between both enzymes. Although we have extensive structural knowledge of the kinase domains of the three IP3-3K isoforms, no structural information is available about the interaction between IP3-3K and Ca2+/CaM. In the present paper we describe the crystal structure of the complex between human Ca2+/CaM and the CaM-binding region of human IP3-3K isoform A (residues 158-183) and propose a model for a complex including the kinase domain. The structure obtained allowed us to identify all of the key residues involved in the interaction, which have been evaluated by site-directed mutagenesis, pull-down and fluorescence anisotropy experiments. The results allowed the identification of a new CaM-binding motif, expanding our knowledge about how CaM interacts with its partners.

  9. Treponema pallidum subsp. pallidum TP0136 Protein Is Heterogeneous among Isolates and Binds Cellular and Plasma Fibronectin via its NH2-Terminal End

    PubMed Central

    Ke, Wujian; Molini, Barbara J.; Lukehart, Sheila A.; Giacani, Lorenzo

    2015-01-01

    Adherence-mediated colonization plays an important role in pathogenesis of microbial infections, particularly those caused by extracellular pathogens responsible for systemic diseases, such as Treponema pallidum subsp. pallidum (T. pallidum), the agent of syphilis. Among T. pallidum adhesins, TP0136 is known to bind fibronectin (Fn), an important constituent of the host extracellular matrix. To deepen our understanding of the TP0136-Fn interaction dynamics, we used two naturally-occurring sequence variants of the TP0136 protein to investigate which region of the protein is responsible for Fn binding, and whether TP0136 would adhere to human cellular Fn in addition to plasma Fn and super Fn as previously reported. Fn binding assays were performed with recombinant proteins representing the two full-length TP0136 variants and their discrete regions. As a complementary approach, we tested inhibition of T. pallidum binding to Fn by recombinant full-length TP0136 proteins and fragments, as well as by anti-TP0136 immune sera. Our results show that TP0136 adheres more efficiently to cellular Fn than to plasma Fn, that the TP0136 NH2-terminal conserved region of the protein is primarily responsible for binding to plasma Fn but that binding sites for cellular Fn are also present in the protein’s central and COOH-terminal regions. Additionally, message quantification studies show that tp0136 is highly transcribed during experimental infection, and that its message level increases in parallel to the host immune pressure on the pathogen, which suggests a possible role for this protein in T. pallidum persistence. In a time where syphilis incidence is high, our data will help in the quest to identify suitable targets for development of a much needed vaccine against this important disease. PMID:25793702

  10. Copper(II) complexes with peptides based on the second cell binding site of fibronectin: metal coordination and ligand exchange kinetics.

    PubMed

    Pizzanelli, Silvia; Forte, Claudia; Pinzino, Calogero; Magrì, Antonio; La Mendola, Diego

    2016-02-07

    Copper(ii) complexes with short peptides based on the second cell binding site of fibronectin, PHSFN and PHSEN, have been characterized by potentiometric, UV-vis, CD, EPR and NMR spectroscopic methods. The histidine imidazole nitrogen is the anchoring site for the metal ion binding. Thermodynamic and spectroscopic evidence is given that the side chain oxygen donor atom of glutamyl residue in Ac-PHSEN-NH2 is also involved in the binding up to physiological pH. To determine ligand exchange kinetic parameters after the imidazole nitrogen anchoring, proton relaxation enhancement NMR data have been collected for the two hydrogen atoms of the imidazole ring in the temperature range 293-315 K at pH 5.2 and globally treated within different kinetic models for ligand exchange. The best fitting model involves two steps. In the first one, which is slow, a water molecule disengages a carbonyl or a carboxylate group coordinated to the metal ion in the complex formed by PHSFN or PHSEN, respectively. This stage is one order of magnitude slower for PHSEN, due to entropic effects. In the second step, which is fast, the complex just formed exchanges with the ligand. In this step, no appreciable differences are found for the two cases examined.

  11. Cloning and characterization of a novel PE_PGRS60 protein (Rv3652) of Mycobacterium tuberculosis H37 Rv exhibit fibronectin-binding property.

    PubMed

    Meena, Laxman S; Meena, Jaishree

    2016-07-01

    The binding of pathogenic bacteria to extracellular matrix components enhances adhesion and invasion of host cells. The host receptor proteins such as fibronectin (Fn) targeted to pathogenic ligands that have clinical importance. In the present study, we cloned, expressed, purified, and identified a novel Fn-binding protein from PE_PGRS60 (Rv3652) of Mycobacterium tuberculosis H37 Rv. The protein product of Rv3652 showed optimum binding efficiency to 10 ng Fn at 0.2 µg purified protein of PE_PGRS60 and 20 ng Fn at 0.2 µg concentrations, respectively. PE_PGRS60 protein (primary sequences) of different pathogenic mycobacterium species retrieved from NCBI exhibited complete homology at the 104 residues on multiple sequence alignment. The primary sequence of protein from H37 Rv was further used to predict cleavage signals. The secondary structure prediction method revealed a number of residues responsible for alpha helices formation and percentage of residues participating in the random coils and extended strands. In addition, online prediction tools such as B- and T-cell epitopes showed the surface probability scale and antigenic propensity scale. The current finding opens new opportunity to mycobacterial survival and pathogenesis research of PE-polymorphic GC-rich repetitive sequences (PE-PGRS) family proteins.

  12. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity.

    PubMed

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun; Nishina, Hiroshi

    2014-01-17

    YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP's functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP's co-activation of TEAD-mediated CTGF transcription.

  13. Fibronectin is a survival factor for differentiated osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Doty, S. B.; Lull, J. C.; Holmuhamedov, E.; Humphries, M. J.; Damsky, C. H.

    1998-01-01

    The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-beta1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-beta1, may affect bone formation, in part by regulating the survival of osteoblasts.

  14. Interferon-induced guanylate-binding proteins lack an N(T)KXD consensus motif and bind GMP in addition to GDP and GTP.

    PubMed

    Cheng, Y S; Patterson, C E; Staeheli, P

    1991-09-01

    The primary structures of interferon (IFN)-induced guanylate-binding proteins (GBPs) were deduced from cloned human and murine cDNAs. These proteins contained only two of the three sequence motifs typically found in GTP/GDP-binding proteins. The N(T)KXD motif, which is believed to confer guanine specificity in other nucleotide-binding proteins, was absent. Nevertheless, the IFN-induced GBPs exhibited a high degree of selectivity for binding to agarose-immobilized guanine nucleotides. An interesting feature of IFN-induced GBPs is that they strongly bound to GMP agarose in addition to GDP and GTP agaroses but failed to bind to ATP agarose and all other nucleotide agaroses tested. Both GTP and GMP, but not ATP, competed for binding of murine GBP-1 to agarose-immobilized GMP. The IFN-induced GBPs thus define a distinct novel family of proteins with GTP-binding activity. We further demonstrate that human and murine cells contain at least two genes encoding IFN-induced GBPs. The cloned murine cDNA codes for GBP-1, an IFN-induced protein previously shown to be absent from mice of Gbp-1b genotype.

  15. Structural domains of heparan sulphate for specific recognition of the C-terminal heparin-binding domain of human plasma fibronectin (HEPII).

    PubMed Central

    Walker, A; Gallagher, J T

    1996-01-01

    Heparan sulphate (HS) is an abundant polysaccharide component of the pericellular domain and is found in most soft tissues and all adherent cells in culture. It interacts with a wide spectrum of proteins including polypeptide growth factors and glycoproteins of the extracellular matrix. These interactions might influence fundamental cellular activities such as adhesion, growth and migration. HS might therefore represent a highly adaptive mechanism by which cells respond to their environment. The present study shows that the interaction between fibroblast HS, metabolically labelled with [3H]glucosamine, and the C-terminal heparin-binding domain of human plasma fibronectin (HEPII), is determined by distinct regions of the polysaccharide chain. By using a very sensitive affinity-chromatography method and specific polysaccharide scission it was shown that the HEPII-binding regions of HS reside within sulphated domains that are resistant to degradation by heparinase III. In addition, optimal binding was achieved with specific heparinase III-resistant fragments of 14-16 monosaccharides in length. The affinity of HS for HEPII was significantly decreased when the polysaccharide was cleaved with heparinase I. Chondroitin sulphate and dermatan sulphate were poor competitive inhibitors of [3H]HS binding to HEPII whereas unlabelled HS and heparin gave a strong inhibitory activity, with heparin being the most potent inhibitor. These findings suggest that the interaction between HEPII and HS is specific and requires extended sequences of seven to eight N-sulphated disaccharides in which a proportion of the iduronate residues are sulphated at C-2. The results have important implications for the functions of HS in cell adhesion and migration. PMID:8760376

  16. Contribution of Sequence Motif, Chromatin State, and DNA Structure Features to Predictive Models of Transcription Factor Binding in Yeast.

    PubMed

    Tsai, Zing Tsung-Yeh; Shiu, Shin-Han; Tsai, Huai-Kuang

    2015-08-01

    Transcription factor (TF) binding is determined by the presence of specific sequence motifs (SM) and chromatin accessibility, where the latter is influenced by both chromatin state (CS) and DNA structure (DS) properties. Although SM, CS, and DS have been used to predict TF binding sites, a predictive model that jointly considers CS and DS has not been developed to predict either TF-specific binding or general binding properties of TFs. Using budding yeast as model, we found that machine learning classifiers trained with either CS or DS features alone perform better in predicting TF-specific binding compared to SM-based classifiers. In addition, simultaneously considering CS and DS further improves the accuracy of the TF binding predictions, indicating the highly complementary nature of these two properties. The contributions of SM, CS, and DS features to binding site predictions differ greatly between TFs, allowing TF-specific predictions and potentially reflecting different TF binding mechanisms. In addition, a "TF-agnostic" predictive model based on three DNA "intrinsic properties" (in silico predicted nucleosome occupancy, major groove geometry, and dinucleotide free energy) that can be calculated from genomic sequences alone has performance that rivals the model incorporating experiment-derived data. This intrinsic property model allows prediction of binding regions not only across TFs, but also across DNA-binding domain families with distinct structural folds. Furthermore, these predicted binding regions can help identify TF binding sites that have a significant impact on target gene expression. Because the intrinsic property model allows prediction of binding regions across DNA-binding domain families, it is TF agnostic and likely describes general binding potential of TFs. Thus, our findings suggest that it is feasible to establish a TF agnostic model for identifying functional regulatory regions in potentially any sequenced genome.

  17. Contribution of Sequence Motif, Chromatin State, and DNA Structure Features to Predictive Models of Transcription Factor Binding in Yeast

    PubMed Central

    Tsai, Zing Tsung-Yeh; Shiu, Shin-Han; Tsai, Huai-Kuang

    2015-01-01

    Transcription factor (TF) binding is determined by the presence of specific sequence motifs (SM) and chromatin accessibility, where the latter is influenced by both chromatin state (CS) and DNA structure (DS) properties. Although SM, CS, and DS have been used to predict TF binding sites, a predictive model that jointly considers CS and DS has not been developed to predict either TF-specific binding or general binding properties of TFs. Using budding yeast as model, we found that machine learning classifiers trained with either CS or DS features alone perform better in predicting TF-specific binding compared to SM-based classifiers. In addition, simultaneously considering CS and DS further improves the accuracy of the TF binding predictions, indicating the highly complementary nature of these two properties. The contributions of SM, CS, and DS features to binding site predictions differ greatly between TFs, allowing TF-specific predictions and potentially reflecting different TF binding mechanisms. In addition, a "TF-agnostic" predictive model based on three DNA “intrinsic properties” (in silico predicted nucleosome occupancy, major groove geometry, and dinucleotide free energy) that can be calculated from genomic sequences alone has performance that rivals the model incorporating experiment-derived data. This intrinsic property model allows prediction of binding regions not only across TFs, but also across DNA-binding domain families with distinct structural folds. Furthermore, these predicted binding regions can help identify TF binding sites that have a significant impact on target gene expression. Because the intrinsic property model allows prediction of binding regions across DNA-binding domain families, it is TF agnostic and likely describes general binding potential of TFs. Thus, our findings suggest that it is feasible to establish a TF agnostic model for identifying functional regulatory regions in potentially any sequenced genome. PMID:26291518

  18. Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides

    PubMed Central

    1986-01-01

    Recent studies have shown that fibronectin and its 140K membrane receptor complex are spatially associated with microfilaments to form cell surface linkage complexes which are thought to mediate adhesive interactions between fibroblasts and their substrata. We examined the regulation of the organization of these cell surface structures in transformed and fibronectin-reconstituted cells as well as in cells treated with a competitive synthetic peptide inhibitor of fibronectin binding to its receptor. Correlative localization experiments with interference reflection microscopy and double-label or triple-label immunofluorescence revealed a concomitant loss of fibronectin, 140K receptor, and alpha-actinin colocalization at cell substratum extracellular matrix contact sites after transformation of chick fibroblasts by wild-type or temperature-sensitive Rous sarcoma viruses (RSV). Western and dot immunoblot analyses established that although similar total quantities of intact 140K molecules were present in the transformed cell cultures, significantly more was released into the culture medium of transformed cells. The 140K molecules on transformed cells were available for interaction with exogenously added fibronectin, which could reconstitute fibronectin-140K linkage complexes. In such fibronectin reconstitution experiments, many cells expressed both fibronectin-140K-actin linkage complexes and RSV pp60src, indicating that the morphological reversion could occur even in the continued presence of RSV transformation. The synthetic peptide Gly-Arg-Gly-Asp-Ser derived from the sequence of the cell-binding region of fibronectin could also prevent the organization of fibronectin-140K linkage complexes. Our results suggest that fibronectin interaction with cells regulates the organization of fibronectin receptor complexes and cytoskeletal components at the cell surface. PMID:3023389

  19. Expression of RNA-binding motif 10 is associated with advanced tumor stage and malignant behaviors of lung adenocarcinoma cancer cells.

    PubMed

    Guan, Guofang; Li, Ranwei; Tang, Wenfang; Liu, Tiecheng; Su, Zhenzhong; Wang, Yan; Tan, Jingjin; Jiang, Shan; Wang, Ke

    2017-03-01

    This study assessed RNA-binding motif 10 expression in lung adenocarcinoma tissues and examined the role and mechanism of RNA-binding motif 10 in the regulation of lung adenocarcinoma malignancy. Lung adenocarcinoma and corresponding adjacent non-tumor lung tissues from 41 patients were subjected to reverse transcription-polymerase chain reaction and Western blot assessment to detect RNA-binding motif 10 expression. Recombinant lentivirus carrying RNA-binding motif 10 complementary DNA was used to infect lung adenocarcinoma cell lines, A549 and H1299 cells. Complementary DNA microarray was used to profile RNA-binding motif 10-regulated genes. Levels of RNA-binding motif 10 messenger RNA and protein were significantly lower in lung adenocarcinoma tissues than those in paired non-tumor tissues (p < 0.001). Reduced RNA-binding motif 10 expression was found to be associated with an advanced tumor stage. RNA-binding motif 10 overexpression inhibited viability and colony formation capacity of lung adenocarcinoma cell lines and induced cell-cycle arrest at G0/G1 phase in A549 cells and at S phase in H1299 cells. Complementary DNA microarray analysis identified 304 upregulated and 386 downregulated genes induced by RNA-binding motif 10 overexpression, which may be involved in cancer, focal adhesion, peroxisome proliferator-activated receptor-regulated gene pathway, cytokine-cytokine receptor interaction, mitogen-activated protein kinase signaling, complement and coagulation cascades, platelet amyloid precursor protein pathway, extracellular matrix-receptor interaction, and small cell lung cancer-related genes. Expression of FGF2, EGFR, WNT5A, NF-κB, and RAP1A was downregulated, whereas expression of AKT2, BIRC3, and JUN was upregulated. RNA-binding motif 10 messenger RNA and protein were reduced in lung adenocarcinoma tissues, and RNA-binding motif 10 overexpression inhibited lung adenocarcinoma cancer cell malignant behavior in vitro. Molecularly, RNA-binding motif

  20. A variety of DNA-binding and multimeric proteins contain the histone fold motif.

    PubMed Central

    Baxevanis, A D; Arents, G; Moudrianakis, E N; Landsman, D

    1995-01-01

    The histone fold motif has previously been identified as a structural feature common to all four core histones and is involved in both histone-histone and histone-DNA interactions. Through the use of a novel motif searching method, a group of proteins containing the histone fold motif has been established. The proteins in this group are involved in a wide variety of functions related mostly to DNA metabolism. Most of these proteins engage in protein-protein or protein-DNA interactions, as do their core histone counterparts. Among these, CCAAT-specific transcription factor CBF and its yeast homologue HAP are two examples of multimeric complexes with different component subunits that contain the histone fold motif. The histone fold proteins are distantly related, with a relatively small degree of absolute sequence similarity. It is proposed that these proteins may share a similar three-dimensional conformation despite the lack of significant sequence similarity. PMID:7651829

  1. Mutation of the Conserved Calcium-Binding Motif in Neisseria gonorrhoeae PilC1 Impacts Adhesion but Not Piliation

    PubMed Central

    Cheng, Yuan; Johnson, Michael D. L.; Burillo-Kirch, Christine; Mocny, Jeffrey C.; Anderson, James E.; Garrett, Christopher K.; Redinbo, Matthew R.

    2013-01-01

    Neisseria gonorrhoeae PilC1 is a member of the PilC family of type IV pilus-associated adhesins found in Neisseria species and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 of Pseudomonas aeruginosa and in PilC1 and PilC2 of Kingella kingae. Genetic analysis of N. gonorrhoeae revealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region in N. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 in K. kingae, but in contrast to the calcium-binding mutant of P. aeruginosa PilY1, an equivalent mutation in N. gonorrhoeae PilC1 produced normal amounts of pili. However, the N. gonorrhoeae PilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function in N. gonorrhoeae. PMID:24002068

  2. Asp residues of βDELSEED-motif are required for peptide binding in the Escherichia coli ATP synthase.

    PubMed

    Ahmad, Zulfiqar; Tayou, Junior; Laughlin, Thomas F

    2015-04-01

    This study demonstrates the requirement of Asp-380 and Asp-386 in the βDELSEED-motif of Escherichia coli ATP synthase for peptide binding and inhibition. We studied the inhibition profiles of wild-type and mutant E. coli ATP synthase in presence of c-terminal amide bound melittin and melittin related peptide. Melittin and melittin related peptide inhibited wild-type ATPase almost completely while only partial inhibition was observed in single mutations with replacement of Asp to Ala, Gln, or Arg. Additionally, very little or no inhibition occurred among double mutants βD380A/βD386A, βD380Q/βD386Q, or βD380R/βD386R signifying that removal of one Asp residue allows limited peptide binding. Partial or substantial loss of oxidative phosphorylation among double mutants demonstrates the functional requirement of βD380 and βD386 Asp residues. Moreover, abrogation of wild-type E. coli cell growth and normal growth of mutant cells in presence of peptides provides strong evidence for the requirement of βDELSEED-motif Asp residues for peptide binding. It is concluded that while presence of one Asp residue may allow partial peptide binding, both Asp residues, βD380 and βD386, are essential for proper peptide binding and inhibition of ATP synthase.

  3. Oxidation-induced Structural Changes of Ceruloplasmin Foster NGR Motif Deamidation That Promotes Integrin Binding and Signaling

    PubMed Central

    Barbariga, Marco; Curnis, Flavio; Spitaleri, Andrea; Andolfo, Annapaola; Zucchelli, Chiara; Lazzaro, Massimo; Magnani, Giuseppe; Musco, Giovanna; Corti, Angelo; Alessio, Massimo

    2014-01-01

    Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface (568NGR) and the other buried in the tertiary structure (962NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. We observed that the exposed 568NGR site can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden 962NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidation-induced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion. PMID:24366863

  4. A DNA-binding protein containing two widely separated zinc finger motifs that recognize the same DNA sequence.

    PubMed

    Fan, C M; Maniatis, T

    1990-01-01

    We have isolated a full-length cDNA clone encoding a protein (PRDII-BF1) that binds specifically to a positive regulatory domain (PRDII) of the human IFN-beta gene promoter, and to a similar sequence present in a number of other promoters and enhancers. The sequence of this protein reveals two novel structural features. First, it is the largest sequence-specific DNA-binding protein reported to date (298 kD). Second, it contains two widely separated sets of C2-H2-type zinc fingers. Remarkably, each set of zinc fingers binds to the same DNA sequence motif with similar affinities and methylation interference patterns. Thus, this protein may act by binding simultaneously to reiterated copies of the same recognition sequence. Although the function of PRDII-BF1 is not known, the level of its mRNA is inducible by serum and virus, albeit with different kinetics.

  5. Inhibitory Member of the Apoptosis-stimulating Proteins of the p53 Family (iASPP) Interacts with Protein Phosphatase 1 via a Noncanonical Binding Motif*

    PubMed Central

    Llanos, Susana; Royer, Christophe; Lu, Min; Bergamaschi, Daniele; Lee, Wen Hwa; Lu, Xin

    2011-01-01

    Although kinase mutations have been identified in various human diseases, much less is known about protein phosphatases. Here, we show that all apoptosis-stimulating proteins of p53 (ASPP) family members can bind protein phosphatase 1 (PP1) via two distinct interacting motifs. ASPP2 interacts with PP1 through an RVXF PP1 binding motif, whereas the inhibitory member of the ASPP family (iASPP) interacts with PP1 via a noncanonical motif (RNYF) that is located within its Src homology 3 domain (SH3). Phe-815 is crucial in mediating iASPP/PP1 interaction, and iASPP(F815A) fails to inhibit the transcriptional and apoptotic function of p53. This study identifies iASPP as a new binding partner of PP1, interacting through a noncanonical PP1 binding motif. PMID:21998301

  6. The N-terminal zinc finger of the erythroid transcription factor GATA-1 binds GATC motifs in DNA.

    PubMed

    Newton, A; Mackay, J; Crossley, M

    2001-09-21

    The mammalian transcription factor GATA-1 is required for normal erythroid and megakaryocytic development. GATA-1 contains two zinc fingers, the C-terminal finger, which is known to bind (A/T)GATA(A/G) motifs in DNA and the N-finger, which is important for interacting with co-regulatory proteins such as Friend of GATA (FOG). We now show that, like the C-finger, the N-finger of GATA-1 is also capable of binding DNA but recognizes distinct sequences with the core GATC. We demonstrate that the GATA-1 N-finger can bind these sequences in vitro and that in cellular assays, GATA-1 can activate promoters containing GATC motifs. Experiments with mutant GATA-1 proteins confirm the importance of the N-finger, as the C-finger is not required for transactivation from GATC sites. Recently four naturally occurring mutations in GATA-1 have been shown to be associated with familial blood disorders. These mutations all map to the N-finger domain. We have investigated the effect of these mutations on the recognition of GATC sites by the N-finger and show that one mutation R216Q abolishes DNA binding, whereas the others have only minor effects.

  7. Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif.

    PubMed

    Zeng, Danyun; Shen, Qingliang; Cho, Jae-Hyun

    2017-02-26

    Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs.

  8. Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

    SciTech Connect

    Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

    2011-11-25

    We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: > We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. > Cre/loxP recombination was used to modify the adenovirus genome. > A targeting ligand present on capsid protein IX was removed or replaced using recombination. > Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

  9. Binding determinants of the small heat shock protein, αB-crystallin: recognition of the ‘IxI' motif

    PubMed Central

    Delbecq, Scott P; Jehle, Stefan; Klevit, Rachel

    2012-01-01

    Small heat shock proteins (sHSPs) play a central role in protein homeostasis under conditions of stress by binding partly unfolded, aggregate-prone proteins and keeping them soluble. Like many sHSPs, the widely expressed human sHSP, αB-crystallin (‘αB'), forms large polydisperse multimeric assemblies. Molecular interactions involved in both sHSP function and oligomer formation remain to be delineated. A growing database of structural information reveals that a central conserved α-crystallin domain (ACD) forms dimeric building blocks, while flanking N- and C-termini direct the formation of larger sHSP oligomers. The most commonly observed inter-subunit interaction involves a highly conserved C-terminal ‘IxI/V' motif and a groove in the ACD that is also implicated in client binding. To investigate the inherent properties of this interaction, peptides mimicking the IxI/V motif of αB and other human sHSPs were tested for binding to dimeric αB-ACD. IxI-mimicking peptides bind the isolated ACD at 22°C in a manner similar to interactions observed in the oligomer at low temperature, confirming these interactions are likely to exist in functional αB oligomers. PMID:23188086

  10. Complete amino acid sequence of BSP-A3 from bovine seminal plasma. Homology to PDC-109 and to the collagen-binding domain of fibronectin.

    PubMed Central

    Seidah, N G; Manjunath, P; Rochemont, J; Sairam, M R; Chrétien, M

    1987-01-01

    Bovine seminal plasma was shown to contain three similar proteins, called BSP-A1, BSP-A2 and BSP-A3. Both BSP-A1 and BSP-A2 were shown to be molecular variants of a recently characterized peptide called PDC-109. They seem to differ only in their degree of glycosylation and otherwise seem to possess an identical amino acid composition. The work in the present paper deals with the complete characterization of the third member of this series, namely BSP-A3. The complete amino acid sequence revealed that it is composed of 115 amino acids and predicts a Mr of 13,403. An analysis of the primary structure of BSP-A3 revealed a high degree of internal homology, with two homologous domains composed of 39 (residues 28-66) and 43 (residues 73-115) amino acids. An exhaustive computer-bank search for the similarity of this sequence to any known protein, or segment thereof, revealed two significant homologies. The first is between PDC-109 and BSP-A3, which is so high that we can confidently predict that both proteins evolved from a single ancestral gene. The collagen-binding domain of bovine fibronectin (type II sequence) was also found to be highly homologous to both BSP-A3 and PDC-109. PMID:3606570

  11. Development of bacterium-based heavy metal biosorbents: Enhanced uptake of cadmium and mercury by Escherichia coli expressing a metal binding motif

    SciTech Connect

    Pazirandeh, M.; Wells, B.M.; Ryan, R.L.

    1998-10-01

    A gene coding for a de novo peptide sequence containing a metal binding motif was chemically synthesized and expressed in Escherichia coli as a fusion with the maltose binding protein. Bacterial cells expressing the metal binding peptide fusion demonstrated enhanced binding of Cd{sup 2+} and Hg{sup 2+} compared to bacterial cells lacking the metal binding peptide. The potential use of genetically engineered bacteria as biosorbents for the removal of heavy metals from wastewaters is discussed.

  12. Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1

    PubMed Central

    Sangadala, Sreedhara; Yoshioka, Katsuhito; Enyo, Yoshio; Liu, Yunshan; Titus, Louisa; Boden, Scott D.

    2014-01-01

    Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquiti-nation and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding

  13. Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1.

    PubMed

    Sangadala, Sreedhara; Yoshioka, Katsuhito; Enyo, Yoshio; Liu, Yunshan; Titus, Louisa; Boden, Scott D

    2014-01-01

    Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquitination and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding

  14. Mass spectrometric characterization of the Campylobacter jejuni adherence factor CadF reveals post-translational processing that removes immunogenicity while retaining fibronectin binding.

    PubMed

    Scott, Nichollas E; Marzook, N Bishara; Deutscher, Ania; Falconer, Linda; Crossett, Ben; Djordjevic, Steven P; Cordwell, Stuart J

    2010-01-01

    Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn-binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2-DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF(24)) and 22 kDa (CadF(22)) mass variants. CadF variants were fully characterized by MALDI-TOF MS and MALDI-MS/MS. These data confirmed that CadF forms re-folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post-translationally. CadF(22) and CadF(24), however, were characterized as N-terminal, membrane-associated polypeptides resulting from cleavage between serine(195) and leucine(196), and glycine(201) and phenylalanine(202), respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full-length CadF (34 kDa), CadF(24), and the deleted C-terminal OmpA domain (14 kDa; CadF(14)) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full-length CadF retained reactivity. Binding assays showed that CadF(24) retained Fn-binding capability, while CadF(14) did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn-binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.

  15. Probing Ca2+-binding capability of viral proteins with the EF-hand motif by grafting approach.

    PubMed

    Zhou, Yubin; Xue, Shenghui; Chen, Yanyi; Yang, Jenny J

    2013-01-01

    Ca(2+) is implicated in almost every step of the life cycle of viruses, including virus entry into host cells, virus replication, virion assembly, maturation, and release. However, due to the lack of prediction algorithms and rigorous validation methods, only limited cases of viral Ca(2+)-binding sites are reported. Here, we introduce a method to predict continuous EF-hand or EF-hand-like motifs in the viral genomes based on their primary sequences. We then introduce a grafting approach, and the use of luminescence resonance energy transfer and Ca(2+) competition assay for experimental verification of predicted Ca(2+)-binding sites. This protocol will be valuable for the prediction and identification of unknown Ca(2+)-binding sites in virus.

  16. Binding motifs in bacterial gene promoters modulate transcriptional effects of global regulators CRP and ArcA

    SciTech Connect

    Leuze, Mike; Karpinets, Tatiana V.; Syed, Mustafa H.; Beliaev, Alex S.; Uberbacher, Edward

    2012-05-30

    Bacterial gene regulation involves transcription factors (TF) that bind to DNA recognition sequences in operon promoters. These recognition sequences, many of which are palindromic, are known as regulatory elements or transcription factor binding sites (TFBS). Some TFs are global regulators that can modulate the expression of hundreds of genes. In this study we examine global regulator half-sites, where a half-site, which we shall call a binding motif (BM), is one half of a palindromic TFBS. We explore the hypothesis that the number of BMs plays an important role in transcriptional regulation, examining empirical data from transcriptional profiling of the CRP and ArcA regulons. We compare the power of BM counts and of full TFBS characteristics to predict induced transcriptional activity. We find that CRP BM counts have a nonlinear effect on CRP-dependent transcriptional activity and predict this activity better than full TFBS quality or location.

  17. The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein

    PubMed Central

    Miranda-Ozuna, Jesús F. T.; Hernández-García, Mar S.; Brieba, Luis G.; Benítez-Cardoza, Claudia G.; Ortega-López, Jaime; González-Robles, Arturo

    2016-01-01

    Triosephosphate isomerase of Trichomonas vaginalis (TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2, that participates in glucose metabolism. TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated TvTIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of TvTIM in T. vaginalis. Affinity ligand assays demonstrated that the recombinant TvTIM1 and TvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r and TvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that TvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis. PMID:27481251

  18. Affinity of the heparin binding motif of Noggin1 to heparan sulfate and its visualization in the embryonic tissues.

    PubMed

    Nesterenko, Alexey M; Orlov, Eugeny E; Ermakova, Galina V; Ivanov, Igor A; Semenyuk, Pavel I; Orlov, Victor N; Martynova, Natalia Y; Zaraisky, Andrey G

    Heparin binding motifs were found in many secreted proteins and it was suggested that they are responsible for retardation of the protein diffusion within the intercellular space due to the binding to heparan sulfate proteoglycanes (HSPG). Here we used synthetic FITC labeled heparin binding motif (HBM peptide) of the Xenopus laevis secreted BMP inhibitor Noggin1 to study its diffusion along the surface of the heparin beads by FRAP method. As a result, we have found out that diffusivity of HBM-labeled FITC was indeed much lesser than those predicted by theoretical calculations even for whole protein of the Noggin size. We also compared by isothermal titration calorimetry the binding affinity of HBM and the control oligolysine peptide to several natural polyanions including heparan sulfate (HS), heparin, the bacterial dextran sulfate and salmon sperm DNA, and demonstrated that HBM significantly exceeds oligolysine peptide in the affinity to HS, heparin and DNA. By contrast, oligolysine peptide bound with higher affinity to dextran sulfate. We speculate that such a difference may ensure specificity of the morphogen binding to HSPG and could be explained by steric constrains imposed by different distribution of the negative charges along a given polymeric molecule. Finally, by using EGFP-HBM recombinant protein we have visualized the natural pattern of the Noggin1 binding sites within the X. laevis gastrula and demonstrated that these sites forms a dorsal-ventral concentration gradient, with a maximum in the dorsal blastopore lip. In sum, our data provide a quantitative basis for modeling the process of Noggin1 diffusion in embryonic tissues, considering its interaction with HSPG.

  19. Genome-wide analysis of ethylene-responsive element binding factor-associated amphiphilic repression motif-containing transcriptional regulators in Arabidopsis.

    PubMed

    Kagale, Sateesh; Links, Matthew G; Rozwadowski, Kevin

    2010-03-01

    The ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif is a transcriptional regulatory motif identified in members of the ethylene-responsive element binding factor, C2H2, and auxin/indole-3-acetic acid families of transcriptional regulators. Sequence comparison of the core EAR motif sites from these proteins revealed two distinct conservation patterns: LxLxL and DLNxxP. Proteins containing these motifs play key roles in diverse biological functions by negatively regulating genes involved in developmental, hormonal, and stress signaling pathways. Through a genome-wide bioinformatics analysis, we have identified the complete repertoire of the EAR repressome in Arabidopsis (Arabidopsis thaliana) comprising 219 proteins belonging to 21 different transcriptional regulator families. Approximately 72% of these proteins contain a LxLxL type of EAR motif, 22% contain a DLNxxP type of EAR motif, and the remaining 6% have a motif where LxLxL and DLNxxP are overlapping. Published in vitro and in planta investigations support approximately 40% of these proteins functioning as negative regulators of gene expression. Comparative sequence analysis of EAR motif sites and adjoining regions has identified additional preferred residues and potential posttranslational modification sites that may influence the functionality of the EAR motif. Homology searches against protein databases of poplar (Populus trichocarpa), grapevine (Vitis vinifera), rice (Oryza sativa), and sorghum (Sorghum bicolor) revealed that the EAR motif is conserved across these diverse plant species. This genome-wide analysis represents the most extensive survey of EAR motif-containing proteins in Arabidopsis to date and provides a resource enabling investigations into their biological roles and the mechanism of EAR motif-mediated transcriptional regulation.

  20. Alteration of the carbohydrate-binding specificity of a C-type lectin CEL-I mutant with an EPN carbohydrate-binding motif.

    PubMed

    Hatakeyama, Tomomitsu; Ishimine, Tomohiro; Baba, Tomohiro; Kimura, Masanari; Unno, Hideaki; Goda, Shuichiro

    2013-07-01

    CEL-I is a Gal/GalNAc-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-recognition domains (CRDs) with the carbohydrate-recognition motif QPD (Gln-Pro- Asp), which is generally known to exist in galactose-specific C-type CRDs. In the present study, a mutant CEL-I with EPN (Glu-Pro-Asn) motif, which is thought to be responsible for the carbohydrate-recognition of mannose-specific Ctype CRDs, was produced in Escherichia coli, and its effects on the carbohydrate-binding specificity were examined using polyamidoamine dendrimer (PD) conjugated with carbohydrates. Although wild-type CEL-I effectively formed complexes with N-acetylgalactosamine (GalNAc)-PD but not with mannose-PD, the mutant CEL-I showed relatively weak but definite affinity for mannose-PD. These results indicated that the QPD and EPN motifs play a significant role in the carbohydrate-recognition mechanism of CEL-I, especially in the discrimination of galactose and mannose. Additional mutations in the recombinant CEL-I binding site may further increase its specificity for mannose, and should provide insights into designing novel carbohydrate-recognition proteins.

  1. New melanocortin 1 receptor binding motif based on the C-terminal sequence of alpha-melanocyte-stimulating hormone.

    PubMed

    Schiöth, Helgi B; Muceniece, Ruta; Mutule, Ilga; Wikberg, Jarl E S

    2006-10-01

    The C-terminal tripeptide of the alpha-melanocyte stimulating hormone (alpha-MSH11-13) possesses strong antiinflammatory activity without known cellular target. In order to better understand the structural requirements for function of such motif, we designed, synthesized and tested out Trp- and Tyr-containing analogues of the alpha-MSH11-13. Seven alpha-MSH11-13 analogues were synthesized and characterized for their binding to the melanocortin receptors recombinantly expressed in insect (Sf9) cells, infected with baculovirus carrying corresponding MC receptor DNA. We also tested these analogues on B16-F1 mouse melanoma cells endogenously expressing the MC1 receptor for binding and for ability to increase cAMP levels as well as on COS-7 cells transfected with the human MC receptors. The data indicate that HS401 (Ac-Tyr-Lys-Pro-Val-NH2) and HS402 (Ac-Lys-Pro-Val-Tyr-NH2) selectively bound to the MC1 receptor and stimulated cAMP generation in a concentration dependent way while the other Tyr- and Trp-containing alpha-MSH11-13 analogues neither bound to MC receptors nor stimulated cAMP. We have thus identified new MC receptor binding motif derived from the C-terminal sequence of alpha-MSH. The tetrapeptides have novel properties as the both act via MC-ergic pathways and also carry the anti-inflammatory alpha-MSH11-13 message sequence.

  2. The NAP motif of activity-dependent neuroprotective protein (ADNP) regulates dendritic spines through microtubule end binding proteins.

    PubMed

    Oz, S; Kapitansky, O; Ivashco-Pachima, Y; Malishkevich, A; Giladi, E; Skalka, N; Rosin-Arbesfeld, R; Mittelman, L; Segev, O; Hirsch, J A; Gozes, I

    2014-10-01

    The NAP motif of activity-dependent neuroprotective protein (ADNP) enhanced memory scores in patients suffering from mild cognitive impairment and protected activities of daily living in schizophrenia patients, while fortifying microtubule (MT)-dependent axonal transport, in mice and flies. The question is how does NAP fortify MTs? Our sequence analysis identified the MT end-binding protein (EB1)-interacting motif SxIP (SIP, Ser-Ile-Pro) in ADNP/NAP and showed specific SxIP binding sites in all members of the EB protein family (EB1-3). Others found that EB1 enhancement of neurite outgrowth is attenuated by EB2, while EB3 interacts with postsynaptic density protein 95 (PSD-95) to modulate dendritic plasticity. Here, NAP increased PSD-95 expression in dendritic spines, which was inhibited by EB3 silencing. EB1 or EB3, but not EB2 silencing inhibited NAP-mediated cell protection, which reflected NAP binding specificity. NAPVSKIPQ (SxIP=SKIP), but not NAPVAAAAQ mimicked NAP activity. ADNP, essential for neuronal differentiation and brain formation in mouse, a member of the SWI/SNF chromatin remodeling complex and a major protein mutated in autism and deregulated in schizophrenia in men, showed similar EB interactions, which were enhanced by NAP treatment. The newly identified shared MT target of NAP/ADNP is directly implicated in synaptic plasticity, explaining the breadth and efficiency of neuroprotective/neurotrophic capacities.

  3. Fibronectin and wound healing.

    PubMed

    Grinnell, F

    1984-01-01

    I have tried to briefly review the evidence (summarized in Table II) indicating that fibronectin is important in cutaneous wound healing. Fibronectin appears to be an important factor throughout this process. It promotes the spreading of platelets at the site of injury, the adhesion and migration of neutrophils, monocytes, fibroblasts, and endothelial cells into the wound region, and the migration of epidermal cells through the granulation tissue. At the level of matrix synthesis, fibronectin appears to be involved both in the organization of the granulation tissue and basement membrane. In terms of tissue remodeling, fibronectin functions as a nonimmune opsonin for phagocytosis of debris by fibroblasts, keratinocytes, and under some circumstances, macrophages. Fibronectin also enhances the phagocytosis of immune-opsonized particles by monocytes, but whether this includes phagocytosis of bacteria remains to be determined. In general, phagocytosis of bacteria has not appeared to involve fibronectin. On the contrary, the presence of fibronectin in the wound bed may promote bacterial attachment and infection. Because of the ease of experimental manipulations, wound healing experiments have been carried out on skin more frequently than other tissues. As a result, the possible role of fibronectin has not been investigated thoroughly in the repair of internal organs and tissues. Nevertheless, it seems reasonable to speculate that fibronectin plays a central role in all wound healing situations. Finally, the wound healing problems of patients with severe factor XIII deficiencies may occur because of their inability to incorporate fibronectin into blood clots.

  4. Definition of an extended MHC class II-peptide binding motif for the autoimmune disease-associated Lewis rat RT1.BL molecule.

    PubMed

    Wauben, M H; van der Kraan, M; Grosfeld-Stulemeyer, M C; Joosten, I

    1997-02-01

    The Lewis rat, an inbred rat strain susceptible to several well-characterized experimental autoimmune diseases, provides a good model to study peptide-mediated immunotherapy. Peptide immunotherapy focussing on the modulation of T cell responses by interfering with TCR-peptide-MHC complex formation requires the elucidation of the molecular basis of TCR-peptide-MHC interactions for an efficient design of modulatory peptides. In the Lewis rat most autoimmune-associated CD4+ T cell responses are MHC class II RT1.BL restricted. In this study, the characteristics of RT1.BL-peptide interactions were explored. A series of substitution analogs of two Lewis rat T cell epitopes was examined in a direct peptide-MHC binding assay on isolated RT1.BL molecules. Furthermore, other autoimmune-related as well as non-disease-related T cell epitopes were tested in the binding assay. This has led to the definition of an extended RT1.BL-peptide binding motif. The RT1.BL-peptide binding motif established in this study is the first described rat MHC-peptide binding motif based on direct MHC-peptide binding experiments. To predict good or intermediate RT1.BL binding peptides, T cell epitope search profiles were deduced from this motif. The motif and search profiles will greatly facilitate the prediction of modulatory peptides based on autoimmune-associated T cell epitopes and the identification of target structures in experimental autoimmune diseases in Lewis rats.

  5. Crystal Structure of (+)-[delta]-Cadinene Synthase from Gossypium arboreum and Evolutionary Divergence of Metal Binding Motifs for Catalysis

    SciTech Connect

    Gennadios, Heather A.; Gonzalez, Veronica; Di Costanzo, Luigi; Li, Amang; Yu, Fanglei; Miller, David J.; Allemann, Rudolf K.; Christianson, David W.

    2009-09-11

    (+)-{delta}-Cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we report the X-ray crystal structure of unliganded DCS at 2.4 {angstrom} resolution and the structure of its complex with three putative Mg{sup 2+} ions and the substrate analogue inhibitor 2-fluorofarnesyl diphosphate (2F-FPP) at 2.75 {angstrom} resolution. These structures illuminate unusual features that accommodate the trinuclear metal cluster required for substrate binding and catalysis. Like other terpenoid cyclases, DCS contains a characteristic aspartate-rich D{sup 307}DTYD{sup 311} motif on helix D that interacts with Mg{sub A}{sup 2+} and Mg{sub C}{sup 2+}. However, DCS appears to be unique among terpenoid cyclases in that it does not contain the 'NSE/DTE' motif on helix H that specifically chelates Mg{sub B}{sup 2+}, which is usually found as the signature sequence (N,D)D(L,I,V)X(S,T)XXXE (boldface indicates Mg{sub B}{sup 2+} ligands). Instead, DCS contains a second aspartate-rich motif, D{sup 451}DVAE{sup 455}, that interacts with Mg{sub B}{sup 2+}. In this regard, DCS is more similar to the isoprenoid chain elongation enzyme farnesyl diphosphate synthase, which also contains two aspartate-rich motifs, rather than the greater family of terpenoid cyclases. Nevertheless, the structure of the DCS-2F-FPP complex shows that the structure of the trinuclear magnesium cluster is generally similar to that of other terpenoid cyclases despite the alternative Mg{sub B}{sup 2+} binding motif. Analyses of DCS mutants with alanine substitutions in the D{sup 307}DTYD{sup 311} and D{sup 451}DVAE{sup 455} segments reveal the contributions of these segments to catalysis.

  6. Reversibly Bound Chloride in the Atrial Natriuretic Peptide Receptor Hormone Binding Domain: Possible Allosteric Regulation and a Conserved Structural Motif for the Chloride-binding Site

    SciTech Connect

    Ogawa, H.; Qiu, Y; Philo, J; Arakawa, T; Ogata, C; Misono, K

    2010-01-01

    The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.

  7. Characterization of the metal ion binding helix-hairpin-helix motifs in human DNA polymerase beta by X-ray structural analysis.

    PubMed

    Pelletier, H; Sawaya, M R

    1996-10-01

    X-ray crystallographic studies have shown that DNA binding by human polymerase beta (pol beta) occurs primarily through two structurally and sequentially homologous helix-hairpin-helix (HhH) motifs, one in the fingers subdomain and the other in the 8-kDa domain [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996a) Biochemistry 35, 12742-12761]. In that DNA binding by each HhH motif is facilitated by a metal ion, we set out to determine the identity of the metal ion that most likely binds to the HhH motif in vivo. Crystal soaking experiments were performed on human pol beta-DNA cocrystals with Mg2+, Ca2+, Na+, and K+, the four most prevalent metal ions in the cell, and in each case a data set was collected and the resulting structure was refined. Under the conditions tested, the HhH motifs of pol beta have an affinity for these biologically prevalent metal ions in the order Mg2+ < Ca2+ < Na+ < K+, with K+ displaying the strongest binding. Crystals soaked in the presence of Tl+, a commonly used spectroscopic probe for K+, were too X-ray-sensitive to establish the binding behavior of Tl+, but soaking experiments with Ba2+ and Cs+ resulted in relatively stable crystals that gave evidence of metal ion binding in both HhH motifs, confirming that larger monovalent and divalent metal ions are capable of binding to the HhH metal sites. Although Mn2+, which has been categorized as a potent polymerase mutagen, binds to the HhH motifs with a greater affinity than Mg2+, Mn2+ does not bind to the HhH motifs in the presence of equimolar concentrations of Na+. These results suggest that in vivo, where Mn2+ is present only in trace amounts, Mn2+ probably does not have a large effect on DNA binding and may instead manifest a mutagenic effect on pol beta primarily by distorting nucleotide binding or by directly affecting the catalytic step [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996b) Biochemistry 35, 12762-12777]. Crystal

  8. TGF-β1 Induces Polypyrimidine Tract-Binding Protein to Alter Fibroblasts Proliferation and Fibronectin Deposition in Keloid

    PubMed Central

    Jiao, Hu; Dong, Ping; Yan, Li; Yang, Zhigang; Lv, Xiaoyan; Li, Qiuchen; Zong, Xianlei; Fan, Jincai; Fu, Xin; Liu, Xia; Xiao, Ran

    2016-01-01

    Human dermal fibrotic disease keloid has been a clinical challenge because of its tumour-like growth and the lack of effective therapy. Dysregulated alternative splicing events have been demonstrated in tumours and fibrosis. In the current study, for the first time, it was demonstrated that the splicing regulator polypyrimidine tract-binding protein (PTB), which plays a pivotal role in tumour proliferation, invasion and metastasis, is overexpressed in keloid tissues and fibroblasts. Additionally, TGF-β1 upregulated the expressions of PTB and its upstream regulator, C-MYC, in keloid fibroblasts. Furthermore, we suppressed PTB using siRNA in keloid fibroblasts and in a keloid xenograft nude mouse model. PTB knockdown significantly slowed the proliferation of keloid fibroblasts and accelerated the regression of transplanted keloid tissues, which was accompanied by a shift in the alternative splicing of USP5 and RTN4. Moreover, when PTB was suppressed, there was a reduction in excessive deposition of FN1 and COL3A1 in transplanted keloid tissues. However, only FN1 was downregulated in keloid fibroblasts that were cultured in media supplemented with TGF-β1. Our study provides evidence for the role of PTB in keloid pathophysiology and offers a novel therapeutic target for keloids. Most importantly, the role TGF-β1 regulation of PTB may provide new insights into the mechanisms underlying inflammatory cytokine-induced fibrosis. PMID:27897224

  9. The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

    PubMed

    Guzmán Prieto, Ana M; Urbanus, Rolf T; Zhang, Xinglin; Bierschenk, Damien; Koekman, C Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P; Pape, Marieke; Paganelli, Fernanda L; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P A; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

    2015-12-17

    Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

  10. The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets

    PubMed Central

    Guzmán Prieto, Ana M.; Urbanus, Rolf T.; Zhang, Xinglin; Bierschenk, Damien; Koekman, C. Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P.; Pape, Marieke; Paganelli, Fernanda L.; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P. A.; Bonten, Marc J. M.; Willems, Rob J. L.; van Schaik, Willem

    2015-01-01

    Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets. PMID:26675410

  11. The impact of RNA binding motif protein 4-regulated splicing cascade on the progression and metabolism of colorectal cancer cells.

    PubMed

    Liang, Yu-Chih; Lin, Wei-Cheng; Lin, Ying-Ju; Lin, Jung-Chun

    2015-11-10

    Dysregulated splicing of pre-messenger (m)RNA is considered a molecular occasion of carcinogenesis. However, the underlying mechanism is complex and remains to be investigated. Herein, we report that the upregulated miR-92a reduced the RNA-binding motif 4 (RBM4) protein expression, leading to the imbalanced expression of the neuronal polypyrimidine tract-binding (nPTB) protein through alternative splicing-coupled nonsense mediated decay (NMD) mechanism. Increase in nPTB protein enhances the relative level of fibroblast growth factor receptor 2 IIIc (FGFR2) and pyruvate kinase M2 (PKM2) transcripts which contribute to the progression and metabolic signature of CRC cells. Expression profiles of RBM4 and downstream alternative splicing events are consistently observed in cancerous tissues compared to adjacent normal tissues. These results constitute a mechanistic understanding of RBM4 on repressing the carcinogenesis of colorectal cells.

  12. A sequence upstream of canonical PDZ-binding motif within CFTR COOH-terminus enhances NHERF1 interaction.

    PubMed

    Sharma, Neeraj; LaRusch, Jessica; Sosnay, Patrick R; Gottschalk, Laura B; Lopez, Andrea P; Pellicore, Matthew J; Evans, Taylor; Davis, Emily; Atalar, Melis; Na, Chan-Hyun; Rosson, Gedge D; Belchis, Deborah; Milewski, Michal; Pandey, Akhilesh; Cutting, Garry R

    2016-12-01

    The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins, such as sodium/hydrogen exchange regulatory factor-1 (NHERF1). In this study, we explored whether COOH-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, Kd = 1,382 ± 61.1 nM) at concentrations well above physiological levels, estimated at 240 nM from RNA-sequencing and 260 nM by liquid chromatography tandem mass spectrometry in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in coimmunoprecipitations using differently tagged versions of NHERF1. Deletion and alanine mutagenesis revealed that a six-amino acid sequence (1417)EENKVR(1422) and the terminal (1478)TRL(1480) (PDZ-binding motif) in the COOH-terminus were essential for the enhanced oligomerization of NHERF1. Full-length CFTR stably expressed in Madin-Darby canine kidney epithelial cells fostered NHERF1 oligomerization that was substantially reduced (∼5-fold) on alanine substitution of EEN, KVR, or EENKVR residues or deletion of the TRL motif. Confocal fluorescent microscopy revealed that the EENKVR and TRL sequences contribute to preferential localization of CFTR to the apical membrane. Together, these results indicate that COOH-terminal sequences mediate enhanced NHERF1 interaction and facilitate the localization of CFTR, a property that could be manipulated to stabilize mutant forms of CFTR at the apical surface to maximize the effect of CFTR-targeted therapeutics.

  13. Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1.

    PubMed

    Fessenden, James D; Feng, Wei; Pessah, Isaac N; Allen, P D

    2004-12-17

    The functional relevance of putative Ca(2+) binding motifs previously identified with Ca(2+) overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca(2+)-imaging techniques, [(3)H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [(3)H]ryanodine binding measurements, both Ca(2+) activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [(3)H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca(2+) concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3-5) did not affect functional responses in 1B5 myotubes except that the EC(50) for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [(3)H]ryanodine binding experiments, the Ca(2+)-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.

  14. Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif

    PubMed Central

    Alenton, Rod Russel R.; Koiwai, Keiichiro; Miyaguchi, Kohei; Kondo, Hidehiro; Hirono, Ikuo

    2017-01-01

    C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation. PMID:28374848

  15. Copper-binding tripeptide motif increases potency of the antimicrobial peptide Anoplin via Reactive Oxygen Species generation.

    PubMed

    Libardo, M Daben J; Nagella, Sai; Lugo, Andrea; Pierce, Scott; Angeles-Boza, Alfredo M

    2015-01-02

    Antimicrobial peptides (AMPs) are broad spectrum antimicrobial agents that act through diverse mechanisms, this characteristic makes them suitable starting points for development of novel classes of antibiotics. We have previously reported the increase in activity of AMPs upon addition of the Amino Terminal Copper and Nickel (ATCUN) Binding Unit. Herein we synthesized the membrane active peptide, Anoplin and two ATCUN-Anoplin derivatives and show that the increase in activity is indeed due to the ROS formation by the Cu(II)-ATCUN complex. We found that the ATCUN-Anoplin peptides were up to four times more potent compared to Anoplin alone against standard test bacteria. We studied membrane disruption, and cellular localization and found that addition of the ATCUN motif did not lead to a difference in these properties. When helical content was calculated, we observed that ATCUN-Anoplin had a lower helical composition. We found that ATCUN-Anoplin are able to oxidatively damage lipids in the bacterial membrane and that their activity trails the rate at which ROS is formed by the Cu(II)-ATCUN complexes alone. This study shows that addition of a metal binding tripeptide motif is a simple strategy to increase potency of AMPs by conferring a secondary action.

  16. Link protein hyaluronan-binding motif abrogates CD44-hyaluronan-mediated leukemia-liver cell adhesion.

    PubMed

    Chen, Jing; Li, Na; Li, Gongchu

    2013-05-01

    The liver is a frequent site for the metastasis of cancer cells originating from other sites. Leukemic liver metastasis is associated with poor prognosis. The ligation of CD44 with hyaluronan (HA) has been shown to contribute to the drug resistance of leukemic cells. In this study, a link protein HA-binding motif was genetically fused with enhanced green fluorescence protein (EGFP) to generate an EGFP-L fusion protein. Furthermore, a coculture system was established to investigate the interaction of leukemic cells with liver cells. CD44-positive Kasumi-1, but not CD44-negative HL-60 cells, were observed to adhere to the liver cell line L02. This cell-cell adhesion was significantly blocked by HA, indicating that Kasumi-L02 cell adhesion was mediated by the CD44-HA interaction. Compared to EGFP, EGFP-L fusion protein bound to L02 and BEL7404 liver cells. EGFP-L partially abrogated the Kasumi-L02 adhesion, suggesting that the link protein-binding motif is able to inhibit CD44-HA-mediated leukemia-liver adhesion. These results may help provide insight into novel therapeutic methods for leukemic patients diagnosed with liver metastasis.

  17. Clustered somatic mutations are frequent in transcription factor binding motifs within proximal promoter regions in melanoma and other cutaneous malignancies

    PubMed Central

    Colebatch, Andrew J.; Di Stefano, Leon; Wong, Stephen Q.; Hannan, Ross D.; Waring, Paul M.; Dobrovic, Alexander

    2016-01-01

    Most cancer DNA sequencing studies have prioritized recurrent non-synonymous coding mutations in order to identify novel cancer-related mutations. Although attention is increasingly being paid to mutations in non-coding regions, standard approaches to identifying significant mutations may not be appropriate and there has been limited analysis of mutational clusters in functionally annotated non-coding regions. We sought to identify clustered somatic mutations (hotspot regions across samples) in functionally annotated regions in melanoma and other cutaneous malignancies (cutaneous squamous cell carcinoma, basal cell carcinoma and Merkel cell carcinoma). Sliding window analyses revealed numerous recurrent clustered hotspot mutations in proximal promoters, with some specific clusters present in up to 25% of cases. Mutations in melanoma were clustered within ETS and Sp1 transcription factor binding motifs, had a UV signature and were identified in other cutaneous malignancies. Clinicopathologic correlation and mutation analysis support a causal role for chronic UV irradiation generating somatic mutations in transcription factor binding motifs of proximal promoters. PMID:27611953

  18. Bean yellow dwarf virus RepA, but not rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif.

    PubMed

    Liu, L; Saunders, K; Thomas, C L; Davies, J W; Stanley, J

    1999-04-10

    It has previously been reported that complementary-sense gene products of wheat dwarf virus (WDV), a geminivirus of the genus Mastrevirus that infects monocotyledonous plants, bind to human and maize retinoblastoma (Rb) protein. Rb proteins control cell-cycle progression by sequestering transcription factors required for entry into S-phase, suggesting that the virus modifies the cellular environment to produce conditions suitable for viral DNA replication. Using a yeast two-hybrid assay, we have investigated whether the complementary-sense gene products of bean yellow dwarf virus, a mastrevirus that is adapted to dicotyledonous plants, also bind maize Rb protein. We demonstrate that whereas RepA binds to Rb protein, Rep does not, suggesting that RepA alone regulates host gene expression and progression of cells to S-phase. RepA mutants containing L --> I, C --> S, C --> G, and E --> Q mutations within the consensus Rb protein binding motif LXCXE retained the ability to bind to Rb, but with reduced efficiency. Most notably, the E --> Q mutation reduced binding by approximately 95%. Nonetheless, all LXCXE mutants were able to replicate in tobacco protoplasts and to systemically infect Nicotiana benthamiana and bean, in which they produced wild-type symptoms.

  19. Regulation of fibronectin gene expression in cardiac fibroblasts by scleraxis.

    PubMed

    Bagchi, Rushita A; Lin, Justin; Wang, Ryan; Czubryt, Michael P

    2016-11-01

    The glycoprotein fibronectin is a key component of the extracellular matrix. By interacting with numerous matrix and cell surface proteins, fibronectin plays important roles in cell adhesion, migration and intracellular signaling. Up-regulation of fibronectin occurs in tissue fibrosis, and previous studies have identified the pro-fibrotic factor TGFβ as an inducer of fibronectin expression, although the mechanism responsible remains unknown. We have previously shown that a key downstream effector of TGFβ signaling in cardiac fibroblasts is the transcription factor scleraxis, which in turn regulates the expression of a wide variety of extracellular matrix genes. We noted that fibronectin expression tracked closely with scleraxis expression, but it was unclear whether scleraxis directly regulated the fibronectin gene. Here, we report that scleraxis acts via two E-box binding sites in the proximal human fibronectin promoter to govern fibronectin expression, with the second E-box being both sufficient and necessary for scleraxis-mediated fibronectin expression to occur. A combination of electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that scleraxis interacted to a greater degree with the second E-box. Over-expression or knockdown of scleraxis resulted in increased or decreased fibronectin expression, respectively, and scleraxis null mice presented with dramatically decreased immunolabeling for fibronectin in cardiac tissue sections compared to wild-type controls. Furthermore, scleraxis was required for TGFβ-induced fibronectin expression: TGFβ lost its ability to induce fibronectin expression following scleraxis knockdown. Together, these results demonstrate a novel and required role for scleraxis in the regulation of cardiac fibroblast fibronectin gene expression basally or in response to TGFβ.

  20. The LIMP-2/SCARB2 Binding Motif on Acid β-Glucosidase

    PubMed Central

    Liou, Benjamin; Haffey, Wendy D.; Greis, Kenneth D.; Grabowski, Gregory A.

    2014-01-01

    The acid β-glucosidase (glucocerbrosidase (GCase)) binding sequence to LIMP-2 (lysosomal integral membrane protein 2), the receptor for intracellular GCase trafficking to the lysosome, has been identified. Heterologous expression of deletion constructs, the available GCase crystal structures, and binding and co-localization of identified peptides or mutant GCases were used to identify and characterize a highly conserved 11-amino acid sequence, DSPIIVDITKD, within human GCase. The binding to LIMP-2 is not dependent upon a single amino acid, but the interactions of GCase with LIMP-2 are heavily influenced by Asp399 and the di-isoleucines, Ile402 and Ile403. A single alanine substitution at any of these decreases GCase binding to LIMP-2 and alters its pH-dependent binding as well as diminishing the trafficking of GCase to the lysosome and significantly increasing GCase secretion. Enterovirus 71 also binds to LIMP-2 (also known as SCARB2) on the external surface of the plasma membrane. However, the LIMP-2/SCARB2 binding sequences for enterovirus 71 and GCase are not similar, indicating that LIMP-2/SCARB2 may have multiple or overlapping binding sites with differing specificities. These findings have therapeutic implications for the production of GCase and the distribution of this enzyme that is delivered to various organs. PMID:25202012

  1. SMN2 exon 7 splicing is inhibited by binding of hnRNP A1 to a common ESS motif that spans the 3' splice site.

    PubMed

    Doktor, Thomas Koed; Schroeder, Lisbeth Dahl; Vested, Anne; Palmfeldt, Johan; Andersen, Henriette Skovgaard; Gregersen, Niels; Andresen, Brage Storstein

    2011-02-01

    Spinal Muscular Atrophy is caused by homozygous loss of SMN1 with phenotypic modulation by SMN2. SMN2 expresses only limited amounts of full-length transcript due to skipping of exon 7 caused by disruption of an SF2/ASF binding ESE. Additionally, hnRNP A1 has been reported to inhibit inclusion of SMN2 exon 7. We previously reported high similarity between the sequence spanning the 3' ss of SMN1 and SMN2 exon 7 and an hnRNP A1 binding ESS, which regulates MCAD exon 5 splicing. We show here that this 3' ss motif indeed functions as a crucial hnRNP A1 binding ESS, which inhibits inclusion of SMN1/2 exon 7 and is antagonized by the SMN1 ESE, but not by the inactive SMN2 sequence. Pull-down experiments revealed a specific interaction between hnRNP A1 and the 3' ss AG-dinucleotide, which could be disrupted by mutations shown to improve splicing in reporter minigenes. Genomic analyses revealed that in the human genome, 3' ss matching the SMN1/2 ESS motif region are much less abundant than 3' ss with a disrupted ESS motif. This indicates that this ESS may be a general splicing inhibitory motif, which binds hnRNP A1 and inhibits exon inclusion by binding to 3' ss harboring this ESS motif.

  2. An extended dsRBD with a novel zinc-binding motif mediates nuclear retention of fission yeast Dicer.

    PubMed

    Barraud, Pierre; Emmerth, Stephan; Shimada, Yukiko; Hotz, Hans-Rudolf; Allain, Frédéric H-T; Bühler, Marc

    2011-08-16

    Dicer proteins function in RNA interference (RNAi) pathways by generating small RNAs (sRNAs). Here, we report the solution structure of the C-terminal domain of Schizosaccharomyces pombe Dicer (Dcr1). The structure reveals an unusual double-stranded RNA binding domain (dsRBD) fold embedding a novel zinc-binding motif that is conserved among dicers in yeast. Although the C-terminal domain of Dcr1 still binds nucleic acids, this property is dispensable for proper functioning of Dcr1. In contrast, disruption of zinc coordination renders Dcr1 mainly cytoplasmic and leads to remarkable changes in gene expression and loss of heterochromatin assembly. In summary, our results reveal novel insights into the mechanism of nuclear retention of Dcr1 and raise the possibility that this new class of dsRBDs might generally function in nucleocytoplasmic trafficking and not substrate binding. The C-terminal domain of Dcr1 constitutes a novel regulatory module that might represent a potential target for therapeutic intervention with fungal diseases.

  3. Analysis of immunoreceptor tyrosine-based activation motif (ITAM) binding to ZAP-70 by surface plasmon resonance.

    PubMed

    Vély, F; Nunès, J A; Malissen, B; Hedgecock, C J

    1997-11-01

    The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine-based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six-eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP-70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP-70 to each TCR-associated ITAM. First, we generated protein tyrosine phosphatase-resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F2p) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase-resistant analogs bind ZAP-70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3 zeta 1 ITAM with a non-peptidic linker made up of gamma-amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP-70 SH2 domains.

  4. Examination of the transcription factor NtcA-binding motif by in vitro selection of DNA sequences from a random library.

    PubMed

    Jiang, F; Wisén, S; Widersten, M; Bergman, B; Mannervik, B

    2000-08-25

    A recursive in vitro selection among random DNA sequences was used for analysis of the cyanobacterial transcription factor NtcA-binding motifs. An eight-base palindromic sequence, TGTA-(N(8))-TACA, was found to be the optimal NtcA-binding sequence. The more divergent the binding sequences, compared to this consensus sequence, the lower the NtcA affinity. The second and third bases in each four-nucleotide half of the consensus sequence were crucial for NtcA binding, and they were in general highly conserved. The most frequently occurring sequence in the middle weakly conserved region was similar to that of the NtcA-binding motif of the Anabaena sp. strain PCC 7120 glnA gene, previously known to have high affinity for NtcA. This indicates that the middle sequences were selected for high NtcA affinity. Analysis of natural NtcA-binding motifs showed that these could be classified into two groups based on differences in recognition consensus sequences. It is suggested that NtcA naturally recognizes different DNA-binding motifs, or has differential affinities to these sequences under different physiological conditions.

  5. An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif.

    PubMed

    Doll, T; Meichsner, M; Riederer, B M; Honegger, P; Matus, A

    1993-10-01

    Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid 'core' sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.

  6. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation.

    PubMed

    Cockburn, Darrell; Nielsen, Morten M; Christiansen, Camilla; Andersen, Joakim M; Rannes, Julie B; Blennow, Andreas; Svensson, Birte

    2015-04-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.

  7. SH3 domains of Grb2 adaptor bind to PXpsiPXR motifs within the Sos1 nucleotide exchange factor in a discriminate manner.

    PubMed

    McDonald, Caleb B; Seldeen, Kenneth L; Deegan, Brian J; Farooq, Amjad

    2009-05-19

    Ubiquitously encountered in a wide variety of cellular processes, the Grb2-Sos1 interaction is mediated through the combinatorial binding of nSH3 and cSH3 domains of Grb2 to various sites containing PXpsiPXR motifs within Sos1. Here, using isothermal titration calorimetry, we demonstrate that while the nSH3 domain binds with affinities in the physiological range to all four sites containing PXpsiPXR motifs, designated S1, S2, S3, and S4, the cSH3 domain can only do so at the S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXpsiPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXpsiPXR motifs at S2, S3, and S4 sites, the PXpsiPXR motif at the S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of the cSH3 domain. In striking contrast, these two additional arginine residues augment the binding of the nSH3 domain to the S1 site, but their role is not critical for the recognition of S2, S3, and S4 sites. Site-directed mutagenesis suggests that the two additional arginine residues flanking the PXpsiPXR motif at the S1 site contribute to free energy of binding via the formation of salt bridges with specific acidic residues in SH3 domains. Molecular modeling is employed to project these novel findings into the 3D structures of SH3 domains in complex with a peptide containing the PXpsiPXR motif and flanking arginine residues at the S1 site. Taken together, this study furthers our understanding of the assembly of a key signaling complex central to cellular machinery.

  8. SH3 Domains of Grb2 Adaptor Bind to PXψPXR Motifs Within the Sos1 Nucleotide Exchange Factor in a Discriminate Manner†

    PubMed Central

    McDonald, Caleb B.; Seldeen, Kenneth L.; Deegan, Brian J.; Farooq, Amjad

    2009-01-01

    Ubiquitously encountered in a wide variety of cellular processes, the Grb2-Sos1 interaction is mediated through the combinatorial binding of nSH3 and cSH3 domains of Grb2 to various sites containing PXψPXR motifs within Sos1. Here, using isothermal titration calorimetry, we demonstrate that while the nSH3 domain binds with affinities in the physiological range to all four sites containing PXψPXR motifs, designated S1, S2, S3 and S4, the cSH3 domain can only do so at S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXψPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXψPXR motifs at S2, S3 and S4 sites, the PXψPXR motif at S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of cSH3 domain. In striking contrast, these two additional arginine residues augment the binding of nSH3 domain to S1 site but their role is not critical for the recognition of S2, S3 and S4 sites. Site-directed mutagenesis suggests that the two additional arginine residues flanking the PXψPXR motif at S1 site contribute to free energy of binding via the formation of salt bridges with specific acidic residues in SH3 domains. Molecular modeling is employed to project these novel findings into the 3D structures of SH3 domains in complex with a peptide containing the PXψPXR motif and flanking arginine residues at S1 site. Taken together, this study furthers our understanding of the assembly of a key signaling complex central to cellular machinery. PMID:19323566

  9. Thermodynamic characterization of the DmsD binding site for the DmsA twin-arginine motif.

    PubMed

    Winstone, Tara M L; Turner, Raymond J

    2015-03-24

    The system specific chaperone DmsD interacts with the twin-arginine leader peptide of its substrate, DmsA, allowing for proper folding and assembly of the DmsA catalytic subunit of dimethyl sulfoxide reductase prior to translocation by the twin-arginine translocase. DmsD residues important for binding the complete 45-amino acid sequence of the DmsA leader (DmsAL) peptide were previously identified and found to cluster in a pocket of the DmsD structure. In this study, we have utilized isothermal titration calorimetry (ITC) to determine the dissociation constant and thermodynamic parameters of 15 single-substitution DmsD variant proteins and a synthetic DmsAL peptide consisting of 27 amino acids (DmsAL₁₅₋₄₁). The stoichiometry values were determined via ITC, and the multimeric compositions of the DmsD variants in the absence and presence of peptide were characterized via size exclusion chromatography and native polyacrylamide gel electrophoresis. An up to 4-fold change in affinity was observed for DmsD variant proteins relative to that of wild-type DmsD, and variation of the entropic contribution to binding divided the binding site into two clusters: residues with either more or less favorable entropy. Substitution of hydrophobic residues along one helix face (helix 5) or prolines found on adjacent loops caused reduced binding affinity because of the increased entropic cost, which suggests that the twin-arginine motif of the DmsAL peptide binds to a preformed site on DmsD. Most DmsD variants were more than 90% monomeric in solution and bound a single peptide per protein molecule. The DmsD variant with the largest dimer population showed increased affinity and induced the formation of tetramers in the presence of peptide, suggesting that dimeric DmsD or an alternatively folded form of DmsD may play an as yet undefined role in binding.

  10. Selective Targeting of G-Quadruplex Structures by a Benzothiazole-Based Binding Motif.

    PubMed

    Buchholz, Ina; Karg, Beatrice; Dickerhoff, Jonathan; Sievers-Engler, Adrian; Lämmerhofer, Michael; Weisz, Klaus

    2017-03-09

    A benzothiazole derivative was identified as potent ligand for DNA G-quadruplex structures. Fluorescence titrations revealed selective binding to quadruplexes of different topologies including parallel, antiparallel and (3+1) hybrid structures. The parallel c-MYC sequence was found to constitute the preferred target with dissociation constants in the micromolar range. Binding of the benzothiazole-based ligand to c-MYC was structurally and thermodynamically characterized in detail by employing a comprehensive set of spectroscopic and calorimetric techniques. Job plot analyses and mass spectral data indicate non-cooperative ligand binding to form 1:1 and 2:1 complex stoichiometries. Whereas stacking interactions are suggested by optical methods, NMR chemical shift perturbations also indicate significant rearrangements of both 5'- and 3'-flanking sequences upon ligand binding. Additional isothermal calorimetry studies yield a thermodynamic profile of the ligand-quadruplex association and reveal enthalpic contributions to be the major driving force for binding. The structural and thermodynamic information obtained in the present work provides the basis for the rational development of benzothiazole derivatives as promising quadruplex binding agents.

  11. IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

    SciTech Connect

    Kwasnicka-Crawford, Dorota A. . E-mail: dakc@yorku.ca; Carson, Andrew R.; Scherer, Stephen W.

    2006-12-01

    The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNA is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions.

  12. One motif to bind them: A small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs.

    PubMed

    Lock, Antonia; Forfar, Rachel; Weston, Cathryn; Bowsher, Leo; Upton, Graham J G; Reynolds, Christopher A; Ladds, Graham; Dixon, Ann M

    2014-12-01

    G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in mammals and facilitate a range of physiological responses triggered by a variety of ligands. GPCRs were thought to function as monomers, however it is now accepted that GPCR homo- and hetero-oligomers also exist and influence receptor properties. The Schizosaccharomyces pombe GPCR Mam2 is a pheromone-sensing receptor involved in mating and has previously been shown to form oligomers in vivo. The first transmembrane domain (TMD) of Mam2 contains a small-XXX-small motif, overrepresented in membrane proteins and well-known for promoting helix-helix interactions. An ortholog of Mam2 in Saccharomyces cerevisiae, Ste2, contains an analogous small-XXX-small motif which has been shown to contribute to receptor homo-oligomerization, localization and function. Here we have used experimental and computational techniques to characterize the role of the small-XXX-small motif in function and assembly of Mam2 for the first time. We find that disruption of the motif via mutagenesis leads to reduction of Mam2 TMD1 homo-oligomerization and pheromone-responsive cellular signaling of the full-length protein. It also impairs correct targeting to the plasma membrane. Mutation of the analogous motif in Ste2 yielded similar results, suggesting a conserved mechanism for assembly. Using co-expression of the two fungal receptors in conjunction with computational models, we demonstrate a functional change in G protein specificity and propose that this is brought about through hetero-dimeric interactions of Mam2 with Ste2 via the complementary small-XXX-small motifs. This highlights the potential of these motifs to affect a range of properties that can be investigated in other GPCRs.

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

    PubMed Central

    Shimoni, L.; Glusker, J. P.

    1995-01-01

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

  14. Binding of the cSH3 domain of Grb2 adaptor to two distinct RXXK motifs within Gab1 docker employs differential mechanisms.

    PubMed

    McDonald, Caleb B; Seldeen, Kenneth L; Deegan, Brian J; Bhat, Vikas; Farooq, Amjad

    2011-01-01

    A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3, and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 3(10) -helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease.

  15. Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

    PubMed Central

    McDonald, Caleb B.; Seldeen, Kenneth L.; Deegan, Brian J.; Bhat, Vikas; Farooq, Amjad

    2010-01-01

    A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3 and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 310-helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease. PMID:21472810

  16. Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly.

    PubMed

    Hung, Liang-Yi; Chen, Hua-Ling; Chang, Ching-Wen; Li, Bor-Ran; Tang, Tang K

    2004-06-01

    We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.

  17. THE HEPARIN-BINDING DOMAIN AND V REGION OF FIBRONECTIN REGULATE APOPTOSIS BY SUPPRESSION OF P53 AND C-MYC IN HUMAN PRIMARY CELLS

    EPA Science Inventory

    In apoptosis the tumor suppressor p53 and oncogene c-myc, are usually upregulated. However, we report here an alternate pathway of regulation that is triggered by inflammatory-associated matrix fragments of fibronectin (FN) and leads to apoptosis. It is mediated by transcriptio...

  18. Ring A of nukacin ISK-1: a lipid II-binding motif for type-A(II) lantibiotic.

    PubMed

    Islam, Mohammad R; Nishie, Mami; Nagao, Jun-ichi; Zendo, Takeshi; Keller, Sandro; Nakayama, Jiro; Kohda, Daisuke; Sahl, Hans-Georg; Sonomoto, Kenji

    2012-02-29

    Ring A of nukacin ISK-1, which is also present in different type-A(II) lantibiotics, resembles a lipid II-binding motif (TxS/TxD/EC, x denotes undefined residues) similar to that present in mersacidin (type-B lantibiotics), which suggests that nukacin ISK-1 binds to lipid II as a docking molecule. Results from our experiments on peptidoglycan precursor (UDP-MurNAc-pp) accumulation and peptide antagonism assays clearly indicated that nukacin ISK-1 inhibits cell-wall biosynthesis, accumulating lipid II precursor inside the cell, and the peptide activity can be repressed by lipid I and lipid II. Interaction analysis of nukacin ISK-1 and different ring A variants with lipid II revealed that nukacin ISK-1 and nukacin D13E (a more active variant) have a high affinity (K(D) = 0.17 and 0.19 μM, respectively) for lipid II, whereas nukacin D13A (a less active variant) showed a lower affinity, and nukacin C14S (a negative variant lacking the ring A structure) exhibited no interaction. Therefore, on the basis of the structural similarity and positional significance of the amino acids in this region, we concluded that nukacin ISK-1 binds lipid II via its ring A region and may lead to the inhibition of cell-wall biosynthesis.

  19. Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif.

    PubMed

    Rosenbaum, Sabrina; Kreft, Sandra; Etich, Julia; Frie, Christian; Stermann, Jacek; Grskovic, Ivan; Frey, Benjamin; Mielenz, Dirk; Pöschl, Ernst; Gaipl, Udo; Paulsson, Mats; Brachvogel, Bent

    2011-02-18

    Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.

  20. Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport

    PubMed Central

    1995-01-01

    In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo. PMID:7490281

  1. Specific binding of the replication protein of plasmid pPS10 to direct and inverted repeats is mediated by an HTH motif.

    PubMed Central

    García de Viedma, D; Serrano-López, A; Díaz-Orejas, R

    1995-01-01

    The initiator protein of the plasmid pPS10, RepA, has a putative helix-turn-helix (HTH) motif at its C-terminal end. RepA dimers bind to an inverted repeat at the repA promoter (repAP) to autoregulate RepA synthesis. [D. García de Viedma, et al. (1996) EMBO J. in press]. RepA monomers bind to four direct repeats at the origin of replication (oriV) to initiate pPS10 replication This report shows that randomly generated mutations in RepA, associated with defficiencies in autoregulation, map either at the putative HTH motif or in its vicinity. These mutant proteins do not promote pPS10 replication and are severely affected in binding to both the repAP and oriV regions in vitro. Revertants of a mutant that map in the vicinity of the HTH motif have been obtained and correspond to a second amino acid substitution far upstream of the motif. However, reversion of mutants that map in the helices of the motif occurs less frequently, at least by an order of magnitude. All these data indicate that the helices of the HTH motif play an essential role in specific RepA-DNA interactions, although additional regions also seem to be involved in DNA binding activity. Some mutations have slightly different effects in replication and autoregulation, suggesting that the role of the HTH motif in the interaction of RepA dimers or monomers with their respective DNA targets (IR or DR) is not the same. Images PMID:8559664

  2. Identification of the Catalytic Ubiquinone-binding Site of Vibrio cholerae Sodium-dependent NADH Dehydrogenase: A NOVEL UBIQUINONE-BINDING MOTIF.

    PubMed

    Tuz, Karina; Li, Chen; Fang, Xuan; Raba, Daniel A; Liang, Pingdong; Minh, David D L; Juárez, Oscar

    2017-02-17

    The sodium-dependent NADH dehydrogenase (Na(+)-NQR) is a key component of the respiratory chain of diverse prokaryotic species, including pathogenic bacteria. Na(+)-NQR uses the energy released by electron transfer between NADH and ubiquinone (UQ) to pump sodium, producing a gradient that sustains many essential homeostatic processes as well as virulence factor secretion and the elimination of drugs. The location of the UQ binding site has been controversial, with two main hypotheses that suggest that this site could be located in the cytosolic subunit A or in the membrane-bound subunit B. In this work, we performed alanine scanning mutagenesis of aromatic residues located in transmembrane helices II, IV, and V of subunit B, near glycine residues 140 and 141. These two critical glycine residues form part of the structures that regulate the site's accessibility. Our results indicate that the elimination of phenylalanine residue 211 or 213 abolishes the UQ-dependent activity, produces a leak of electrons to oxygen, and completely blocks the binding of UQ and the inhibitor HQNO. Molecular docking calculations predict that UQ interacts with phenylalanine 211 and pinpoints the location of the binding site in the interface of subunits B and D. The mutagenesis and structural analysis allow us to propose a novel UQ-binding motif, which is completely different compared with the sites of other respiratory photosynthetic complexes. These results are essential to understanding the electron transfer pathways and mechanism of Na(+)-NQR catalysis.

  3. Identification of the bioactive and consensus peptide motif from Momordica charantia insulin receptor-binding protein.

    PubMed

    Lo, Hsin-Yi; Li, Chia-Cheng; Ho, Tin-Yun; Hsiang, Chien-Yun

    2016-08-01

    Many food bioactive peptides with diverse functions have been discovered by studying plant proteins. We have previously identified a 68-residue insulin receptor (IR)-binding protein (mcIRBP) from Momordica charantia that exhibits hypoglycemic effects in mice via interaction with IR. By in vitro digestion, we found that mcIRBP-19, spanning residues 50-68 of mcIRBP, enhanced the binding of insulin to IR, stimulated the phosphorylation of PDK1 and Akt, induced the expression of glucose transporter 4, and stimulated both the uptake of glucose in cells and the clearance of glucose in diabetic mice. Furthermore, mcIRBP-19 homologs were present in various plants and shared similar β-hairpin structures and IR kinase-activating abilities to mcIRBP-19. In conclusion, our findings suggested that mcIRBP-19 is a blood glucose-lowering bioactive peptide that exhibits IR-binding potentials. Moreover, we newly identified novel IR-binding bioactive peptides in various plants which belonged to different taxonomic families.

  4. Fibronectin is not Present in the Focal Adhesions Formed between Normal Cultured Fibroblasts and Their Substrata

    NASA Astrophysics Data System (ADS)

    Chen, Wen-Tien; Singer, S. J.

    1980-12-01

    Fibronectin is an extracellular matrix protein that has been implicated in the spreading and adhesion of cultured fibroblasts to their substrata. In this paper, double immunoelectron microscopic labeling experiments for fibronectin and for concanavalin A-binding proteins on the cell surface were carried out on ultrathin frozen sections of cultures of embryonic chicken heart fibroblasts. On cross sections through the focal adhesions of the cell to the substratum there was substantial labeling for concanavalin A-binding proteins but no detectable labeling for fibronectin, whereas both the binding proteins and fibronectin were extensively labeled elsewhere on the cell surface and substratum. These results demonstrate that fibronectin is not present within the sites of focal adhesions. Therefore, the functions of fibronectin in cell spreading and adhesion are not directly mediated through its binding at focal adhesion sites. An alternative model is presented which can account for such fibronectin functions.

  5. Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif

    SciTech Connect

    Carlow, D.A.; Teh, H.S.; Marth, J.

    1995-02-15

    In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4{sup +}8{sup +} thymocytes upon TCR cross-linking. In contrast, expression of Tgtp in peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of {approx}50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation. 51 refs., 6 figs.

  6. A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

    PubMed Central

    2012-01-01

    Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369), containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds. PMID:23190769

  7. Supramolecular Polymers with Multiple Types of Binding Motifs: From Fundamental Studies to Multifunctional Materials

    DTIC Science & Technology

    2015-07-10

    the goal of preparing multi-responsive polymer actuators, we have incorporated liquid crystalline metal-binding Bip monomers into polymeric networks...SECURITY CLASSIFICATION OF: This research project is focused on the development and investigation of a new class of multi-stimuli-responsive polymers ...studies metallo and hydrogen bonded supramolecular polymers that exhibit defect healing characteristics and multi- 1. REPORT DATE (DD-MM-YYYY) 4

  8. Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets

    PubMed Central

    Davies, Douglas R.; Gelinas, Amy D.; Zhang, Chi; Rohloff, John C.; Carter, Jeffrey D.; O’Connell, Daniel; Waugh, Sheela M.; Wolk, Steven K.; Mayfield, Wesley S.; Burgin, Alex B.; Edwards, Thomas E.; Stewart, Lance J.; Gold, Larry; Janjic, Nebojsa; Jarvis, Thale C.

    2012-01-01

    Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional protein-binding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets. PMID:23139410

  9. Binding motifs of CBP2 a potential cell surface target for carcinoma cells.

    PubMed

    Sauk, J J; Coletta, R D; Norris, K; Hebert, C

    2000-05-01

    Previously we have shown (Hebert et al. [1999] J. Cell Biochem. 73:248-258) that among many cell lines the CBP2 gene product, Hsp47, eludes its retention receptor, erd2P, resulting in the appearance of Hsp47 on the cell surface associated with the tetraspanin protein CD9. Since Hsp47 possesses a highly restricted binding cleft, random peptide display libraries were used to characterize peptides binding to Hsp47 and then to target this protein on carcinoma cell lines in vitro. Comparison of the clones obtained from panning revealed little specific homology based on sequence alone. To determine whether carcinoma cells expressing Hsp47 could selectively take up the selected bacteriophages, traditional immunofluorescence and confocal microscopy were employed. These studies revealed that phage-displaying Hsp47 binding peptides bound to cell lines expressing Hsp47 and that the peptides were rapidly taken up to a location coincident with Hsp47 staining. These observations were confirmed by cytometric analyses. These data indicate that CBP2 product may provide a molecular target for chemotherapy and/or imaging of malignancies.

  10. LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance.

    PubMed

    Wilbert, Melissa L; Huelga, Stephanie C; Kapeli, Katannya; Stark, Thomas J; Liang, Tiffany Y; Chen, Stella X; Yan, Bernice Y; Nathanson, Jason L; Hutt, Kasey R; Lovci, Michael T; Kazan, Hilal; Vu, Anthony Q; Massirer, Katlin B; Morris, Quaid; Hoon, Shawn; Yeo, Gene W

    2012-10-26

    LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.

  11. LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance

    PubMed Central

    Wilbert, Melissa L.; Huelga, Stephanie C.; Kapeli, Katannya; Stark, Thomas J.; Liang, Tiffany Y.; Chen, Stella X.; Yan, Bernice Y.; Nathanson, Jason L.; Hutt, Kasey R.; Lovci, Michael T.; Kazan, Hilal; Vu, Anthony Q; Massirer, Katlin B.; Morris, Quaid; Hoon, Shawn; Yeo, Gene W.

    2012-01-01

    SUMMARY LIN28 is a conserved RNA binding protein implicated in pluripotency, reprogramming and oncogenesis. Previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through cross-linking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28 binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions. PMID:22959275

  12. Structure prediction of LDLR-HNP1 complex based on docking enhanced by LDLR binding 3D motif.

    PubMed

    Esmaielbeiki, Reyhaneh; Naughton, Declan P; Nebel, Jean-Christophe

    2012-04-01

    Human antimicrobial peptides (AMPs), including defensins, have come under intense scrutiny owing to their key multiple roles as antimicrobial agents. Not only do they display direct action on microbes, but also recently they have been shown to interact with the immune system to increase antimicrobial activity. Unfortunately, since mechanisms involved in the binding of AMPs to mammalian cells are largely unknown, their potential as novel anti-infective agents cannot be exploited yet. Following the reported interaction of Human Neutrophil Peptide 1 dimer (HNP1) with a low density lipoprotein receptor (LDLR), a computational study was conducted to discover their putative mode of interaction. State-of-the-art docking software produced a set of LDLR-HNP1 complex 3D models. Creation of a 3D motif capturing atomic interactions of the LDLR binding interface allowed selection of the most plausible configurations. Eventually, only two models were in agreement with the literature. Binding energy estimations revealed that only one of them is particularly stable, but also interaction with LDLR weakens significantly bonds within the HNP1 dimer. This may be significant since it suggests a mechanism for internalisation of HNP1 in mammalian cells. In addition to a novel approach for complex structure prediction, this study proposes a 3D model of the LDLR-HNP1 complex which highlights the key residues which are involved in the interactions. The putative identification of the receptor binding mechanism should inform the future design of synthetic HNPs to afford maximum internalisation, which could lead to novel anti-infective drugs.

  13. Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

    PubMed Central

    Sato, Seiichi; Zhao, Yingshe; Imai, Misa; Simister, Philip C.; Feller, Stephan M.; Trackman, Philip C.; Kirsch, Kathrin H.; Sonenshein, Gail E.

    2013-01-01

    The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the “Cbl-interacting protein of 85-kDa” (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells. PMID

  14. The structure of the translational initiation factor IF1 from E.coli contains an oligomer-binding motif.

    PubMed Central

    Sette, M; van Tilborg, P; Spurio, R; Kaptein, R; Paci, M; Gualerzi, C O; Boelens, R

    1997-01-01

    The structure of the translational initiation factor IF1 from Escherichia coli has been determined with multidimensional NMR spectroscopy. Using 1041 distance and 78 dihedral constraints, 40 distance geometry structures were calculated, which were refined by restrained molecular dynamics. From this set, 19 structures were selected, having low constraint energy and few constraint violations. The ensemble of 19 structures displays a root-mean-square deviation versus the average of 0.49 A for the backbone atoms and 1.12 A for all atoms for residues 6-36 and 46-67. The structure of IF1 is characterized by a five-stranded beta-barrel. The loop connecting strands three and four contains a short 3(10) helix but this region shows considerably higher flexibility than the beta-barrel. The fold of IF1 is very similar to that found in the bacterial cold shock proteins CspA and CspB, the N-terminal domain of aspartyl-tRNA synthetase and the staphylococcal nuclease, and can be identified as the oligomer-binding motif. Several proteins of this family are nucleic acid-binding proteins. This suggests that IF1 plays its role in the initiation of protein synthesis by nucleic acid interactions. Specific changes of NMR signals of IF1 upon titration with 30S ribosomal subunit identifies several residues that are involved in the interaction with ribosomes. PMID:9135158

  15. Identification of a positively evolving putative binding region with increased variability in posttranslational motifs in zonadhesin MAM domain 2.

    PubMed

    Herlyn, Holger; Zischler, Hans

    2005-10-01

    Positive selection has been shown to be pervasive in sex-related proteins of many metazoan taxa. However, we are only beginning to understand molecular evolutionary processes on the lineage to humans. To elucidate the evolution of proteins involved in human reproduction, we studied the sequence evolution of MAM domains of the sperm-ligand zonadhesin in respect to single amino acid sites, solvent accessibility, and posttranslational modification. GenBank-data were supplemented by new cDNA-sequences of a representative non-human primate panel. Solvent accessibility predictions identified a probably exposed fragment of 30 amino acids belonging to MAM domain 2 (i.e., MAM domain 3 in mouse). The fragment is characterized by significantly increased rate of positively selected amino acid sites and exhibits high variability in predicted posttranslational modification, and, thus, might represent a binding region in the mature protein. At the same time, there is a significant coincidence of positively selected amino acid sites and non-conserved posttranslational motifs. We conclude that the binding specificity of zonadhesin MAM domains, especially of the presumed epitope, is achieved by positive selection at the level of single amino acid sites and posttranslational modifications, respectively.

  16. The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif

    PubMed Central

    Awad, Rida; Marion, Sévajol; Isabel, Ayala; Anne, Chouquet; Philippe, Frachet; Pierre, Gans; Jean-Baptiste, Reiser; Jean-Philippe, Kleman

    2015-01-01

    Eukaryotic EnguLfment and cell MOtility (ELMO) proteins form an evolutionary conserved family of regulators involved in small GTPase dependent actin remodeling processes that regulates the guanine exchange factor activity of some of the Downstream Of CrK (DOCK) family members. Gathered data strongly suggest that DOCK activation by ELMO and the subsequent signaling result from a subtle balance in the binding of partners to ELMO. Among its putative upward modulators, the Hematopoietic cell kinase (Hck), a member of the Src kinase superfamily, has been identified as a binding partner and a specific tyrosine kinase for ELMO1. Indeed, Hck is implicated in distinct molecular signaling pathways governing phagocytosis, cell adhesion, and migration of hematopoietic cells. Although ELMO1 has been shown to interact with the regulatory Src Homology 3 (SH3) domain of Hck, no direct evidence indicating the mode of interaction between Hck and ELMO1 have been provided in the literature. In the present study, we report convergent pieces of evidence that demonstrate the specific interaction between the SH3 domain of Hck and the polyproline motif of ELMO1. Our results also suggest that the tyrosine-phosphorylation state of ELMO1 tail might act as a putative modulator of Hck kinase activity towards ELMO1 that in turn participates in DOCK180 activation and further triggers subsequent signaling towards actin remodeling. PMID:25737835

  17. Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes.

    PubMed

    Varela-Eirin, Marta; Varela-Vazquez, Adrian; Rodríguez-Candela Mateos, Marina; Vila-Sanjurjo, Anton; Fonseca, Eduardo; Mascareñas, José L; Eugenio Vázquez, M; Mayan, Maria D

    2017-04-01

    Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between virtually all cells in solid tissues and are essential for functional coordination by enabling the direct transfer of small signalling molecules, metabolites, ions, and electrical signals from cell to cell. Several studies have revealed diverse channel-independent functions of Cxs, which include the control of cell growth and tumourigenicity. Connexin43 (Cx43) is the most widespread Cx in the human body. The myriad roles of Cx43 and its implication in the development of disorders such as cancer, inflammation, osteoarthritis and Alzheimer's disease have given rise to many novel questions. Several RNA- and DNA-binding motifs were predicted in the Cx43 and Cx26 sequences using different computational methods. This review provides insights into new, ground-breaking functions of Cxs, highlighting important areas for future work such as transfer of genetic information through extracellular vesicles. We discuss the implication of potential RNA- and DNA-binding domains in the Cx43 and Cx26 sequences in the cellular communication and control of signalling pathways.

  18. Despite a Conserved Cystine Knot Motif, Different Cyclotides Have Different Membrane Binding Modes

    PubMed Central

    Wang, Conan K.; Colgrave, Michelle L.; Ireland, David C.; Kaas, Quentin; Craik, David J.

    2009-01-01

    Abstract Cyclotides are cyclic proteins produced by plants for defense against pests. Because of their remarkable stability and diverse bioactivities, they have a range of potential therapeutic applications. The bioactivities of cyclotides are believed to be mediated through membrane interactions. To determine the structural basis for the biological activity of the two major subfamilies of cyclotides, we determined the conformation and orientation of kalata B2 (kB2), a Möbius cyclotide, and cycloviolacin O2 (cO2), a bracelet cyclotide, bound to dodecylphosphocholine micelles, using NMR spectroscopy in the presence and absence of 5- and 16-doxylstearate relaxation probes. Analysis of binding curves using the Langmuir isotherm indicated that cO2 and kB2 have association constants of 7.0 × 103 M−1 and 6.0 × 103 M−1, respectively, consistent with the notion that they are bound near the surface, rather than buried deeply within the micelle. This suggestion is supported by the selective broadening of micelle-bound cyclotide NMR signals upon addition of paramagnetic Mn ions. The cyclotides from the different subfamilies exhibited clearly different binding orientations at the micelle surface. Structural analysis of cO2 confirmed that the main element of the secondary structure is a β-hairpin centered in loop 5. A small helical turn is present in loop 3. Analysis of the surface profile of cO2 shows that a hydrophobic patch stretches over loops 2 and 3, in contrast to the hydrophobic patch of kB2, which predominantly involves loops 2 and 5. The different location of the hydrophobic patches in the two cyclotides explains their different binding orientations and provides an insight into the biological activities of cyclotides. PMID:19720036

  19. A Conserved Phenylalanine of Motif IV in Superfamily 2 Helicases Is Required for Cooperative, ATP-Dependent Binding of RNA Substrates in DEAD-Box Proteins▿ †

    PubMed Central

    Banroques, Josette; Cordin, Olivier; Doère, Monique; Linder, Patrick; Tanner, N. Kyle

    2008-01-01

    We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-π interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding. PMID:18332124

  20. A large displacement of the SXN motif of Cys115-modified penicillin-binding protein 5 from Escherichia coli

    PubMed Central

    2005-01-01

    Penicillin-binding proteins (PBPs), which are the lethal targets of β-lactam antibiotics, catalyse the final stages of peptidoglycan biosynthesis of the bacterial cell wall. PBP 5 of Escherichia coli is a D-alanine CPase (carboxypeptidase) that has served as a useful model to elucidate the catalytic mechanism of low-molecular-mass PBPs. Previous studies have shown that modification of Cys115 with a variety of reagents results in a loss of CPase activity and a large decrease in the rate of deacylation of the penicilloyl–PBP 5 complex [Tamura, Imae and Strominger (1976) J. Biol. Chem. 251, 414–423; Curtis and Strominger (1978) J. Biol. Chem. 253, 2584–2588]. The crystal structure of wild-type PBP 5 in which Cys115 fortuitously had formed a covalent adduct with 2-mercaptoethanol was solved at 2.0 Å (0.2 nm) resolution, and these results provide a structural rationale for how thiol-directed reagents lower the rate of deacylation. When compared with the structure of the unmodified wild-type enzyme, a major change in the architecture of the active site is observed. The two largest differences are the disordering of a loop comprising residues 74–90 and a shift in residues 106–111, which results in the displacement of Ser110 of the SXN active-site motif. These results support the developing hypothesis that the SXN motif of PBP 5, and especially Ser110, is intimately involved in the catalytic mechanism of deacylation. PMID:16038617

  1. Guidelines for choosing molecular "alligator clip" binding motifs in electron transport devices

    NASA Astrophysics Data System (ADS)

    Reuter, Matthew G.; Seideman, Tamar; Ratner, Mark A.

    2011-04-01

    We employ a one-electron, tight-binding model of an electrode-molecule-electrode junction to explore the fundamental relationship between adsorption geometry and electron transport, producing exact results (within this model). By varying the chemisorption location (e.g., atop a surface atom or in a hollow site between surface atoms) and the molecule-electrode coupling, we find that the largest currents are realized when the molecule (i) is highly coordinated by the surface and (ii) has favorable overlap with electrode states near the Fermi level. We also show the importance of electrode-induced molecular level shifting for certain adsorption geometries, which can cause molecular levels far from the Fermi level to conduct better than those near the Fermi level. Since all of these factors are greatly influenced by the chemical moiety used to link the molecule to an electrode, these results present a set of guidelines to help choose "alligator clips" for molecular electronic devices.

  2. A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus.

    PubMed Central

    Olabarría, G; Carrascosa, J L; de Pedro, M A; Berenguer, J

    1996-01-01

    There is experimental evidence to suggest that the 100-kDa S-layer protein from Thermus thermophilus HB8 binds to the peptidoglycan cell wall. This property could be related to the presence of a region (SLH) of homology with other S-layer proteins and extracellular enzymes (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). By using specific monoclonal antibodies, we show that similar regions are present in different members of the Deinococcus-Thermus phylogenetic group. To analyze the role that the SLH domain plays in vivo and in vitro in T. thermophilus, we have obtained a mutant form (slpA.X) of the S-layer gene (slpA) in which the SLH domain was deleted. The slpA.X gene was inserted into the chromosome of the thermophile by gene replacement, resulting in a mutant which expressed a major membrane protein with the size expected from the construction (90 kDa). This protein was identified as the product of slpA.X by its differential reaction with monoclonal antibodies. Mutants expressing the SlpA.X protein grow as groups of cells, surrounded by a common external envelope of trigonal symmetry that contains the SlpA.X protein as a main component, thus showing the inability of the SLH-defective protein to attach to the underlying material in vivo. In addition, averaged images of SlpA.X-rich fractions showed a regular arrangement, identical to that built up by the wild-type (SlpA) protein in the absence of peptidoglycan. Finally, we demonstrate by Western blotting (immunoblotting) the direct role of the SLH domain in the binding of the S-layer of T. thermophilus HB8 to the peptidoglycan layer. PMID:8759836

  3. Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

    PubMed

    Santamaría-Hernando, Saray; Krell, Tino; Ramos-González, María-Isabel

    2012-01-01

    Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca(2+) coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+) binding with a K(D) of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.

  4. The Ku-binding motif is a conserved module for recruitment and stimulation of non-homologous end-joining proteins

    PubMed Central

    Grundy, Gabrielle J.; Rulten, Stuart L.; Arribas-Bosacoma, Raquel; Davidson, Kathryn; Kozik, Zuzanna; Oliver, Antony W.; Pearl, Laurence H.; Caldecott, Keith W.

    2016-01-01

    The Ku-binding motif (KBM) is a short peptide module first identified in APLF that we now show is also present in Werner syndrome protein (WRN) and in Modulator of retrovirus infection homologue (MRI). We also identify a related but functionally distinct motif in XLF, WRN, MRI and PAXX, which we denote the XLF-like motif. We show that WRN possesses two KBMs; one at the N terminus next to the exonuclease domain and one at the C terminus next to an XLF-like motif. We reveal that the WRN C-terminal KBM and XLF-like motif function cooperatively to bind Ku complexes and that the N-terminal KBM mediates Ku-dependent stimulation of WRN exonuclease activity. We also show that WRN accelerates DSB repair by a mechanism requiring both KBMs, demonstrating the importance of WRN interaction with Ku. These data define a conserved family of KBMs that function as molecular tethers to recruit and/or stimulate enzymes during NHEJ. PMID:27063109

  5. Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function

    PubMed Central

    1984-01-01

    Fibronectin and certain polypeptide regions of this adhesive glycoprotein mediate cell attachment and spreading on various substrates. We explored the theoretical prediction that this adhesive protein could become a competitive inhibitor of fibronectin-mediated processes if present in solution at appropriately high concentrations. Fibronectin function was inhibited by purified plasma fibronectin at 5- 10 mg/ml, by a 75,000-dalton cell-interaction fragment of the protein at 0.5-1 mg/ml, and even by two synthetic peptides containing a conserved, hydrophilic amino acid sequence at 0.1-0.5 mg/ml. Inhibition of fibronectin-dependent cell spreading was dose dependent, noncytotoxic, and reversible. It was competitive in nature, since increased quantities of substrate-adsorbed fibronectin or longer incubation periods decreased the inhibition. A peptide inhibitory for fibronectin-mediated cell spreading also inhibited fibronectin-mediated attachment of cells to type I collagen, but it did not affect concanavalin A-mediated spreading. These results demonstrate the potential of a cell adhesion molecule and its biologically active peptide fragments to act as competitive inhibitors, and they suggest that fibronectin may act by binding to a saturable cell surface receptor. PMID:6736130

  6. ChIP-exo signal associated with DNA-binding motifs provides insight into the genomic binding of the glucocorticoid receptor and cooperating transcription factors

    PubMed Central

    Starick, Stephan R.; Ibn-Salem, Jonas; Jurk, Marcel; Hernandez, Céline; Love, Michael I.; Chung, Ho-Ryun; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H.

    2015-01-01

    The classical DNA recognition sequence of the glucocorticoid receptor (GR) appears to be present at only a fraction of bound genomic regions. To identify sequences responsible for recruitment of this transcription factor (TF) to individual loci, we turned to the high-resolution ChIP-exo approach. We exploited this signal by determining footprint profiles of TF binding at single-base-pair resolution using ExoProfiler, a computational pipeline based on DNA binding motifs. When applied to our GR and the few available public ChIP-exo data sets, we find that ChIP-exo footprints are protein- and recognition sequence-specific signatures of genomic TF association. Furthermore, we show that ChIP-exo captures information about TFs other than the one directly targeted by the antibody in the ChIP procedure. Consequently, the shape of the ChIP-exo footprint can be used to discriminate between direct and indirect (tethering to other DNA-bound proteins) DNA association of GR. Together, our findings indicate that the absence of classical recognition sequences can be explained by direct GR binding to a broader spectrum of sequences than previously known, either as a homodimer or as a heterodimer binding together with a member of the ETS or TEAD families of TFs, or alternatively by indirect recruitment via FOX or STAT proteins. ChIP-exo footprints also bring structural insights and locate DNA:protein cross-link points that are compatible with crystal structures of the studied TFs. Overall, our generically applicable footprint-based approach uncovers new structural and functional insights into the diverse ways of genomic cooperation and association of TFs. PMID:25720775

  7. Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras

    PubMed Central

    Warren, Jeremy G.; Lincoln, James E.; Kirkpatrick, Bruce C.

    2015-01-01

    Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or

  8. Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

    PubMed

    Warren, Jeremy G; Lincoln, James E; Kirkpatrick, Bruce C

    2015-01-01

    Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or

  9. Adhesive and Migratory Effects of Phosphophoryn Are Modulated by Flanking Peptides of the Integrin Binding Motif

    PubMed Central

    Suzuki, Shigeki; Kobuke, Seiji; Haruyama, Naoto; Hoshino, Hiroaki; Kulkarni, Ashok B.; Nishimura, Fusanori

    2014-01-01

    Phosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). Gene duplications in the ancestor dentin matrix protein-1 (DMP-1) genomic sequence created the DSPP gene in toothed animals. PP and DMP-1 are phosphorylated extracellular matrix proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Many SIBLING members have been shown to evoke various cell responses through the integrin-binding Arg-Gly-Asp (RGD) domain; however, the RGD-dependent function of PP is not yet fully understood. We demonstrated that recombinant PP did not exhibit any obvious cell adhesion ability, whereas the simultaneously purified recombinant DMP-1 did. A cell adhesion inhibitory analysis was performed by pre-incubating human osteosarcoma MG63 cells with various PP peptides before seeding onto vitronectin. The results obtained revealed that the incorporation of more than one amino acid on both sides of the PP-RGD domain was unable to inhibit the adhesion of MG63 cells onto vitronectin. Furthermore, the inhibitory activity of a peptide containing the PP-RGD domain with an open carboxyl-terminal side (H-463SDESDTNSESANESGSRGDA482-OH) was more potent than that of a peptide containing the RGD domain with an open amino-terminal side (H-478SRGDASYTSDESSDDDNDSDSH499-OH). This phenomenon was supported by the potent cell adhesion and migration abilities of the recombinant truncated PP, which terminated with Ala482. Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins. Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered. The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin

  10. Fibronectin Aggregation and Assembly

    PubMed Central

    Ohashi, Tomoo; Erickson, Harold P.

    2011-01-01

    The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region (I1–9) is commonly accepted as one of the assembly sites. We previously found that I1–9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that I1–9 bound to the aggregate formed by anastellin and a small FN fragment, III1–2. An engineered disulfide bond in III2, which stabilizes folding, inhibited aggregation, but a disulfide bond in III1 did not. A gelatin precipitation assay showed that I1–9 did not interact with anastellin, III1, III2, III1–2, or several III1–2 mutants including III1–2KADA. (In contrast to previous studies, we found that the III1–2KADA mutant was identical in conformation to wild-type III1–2.) Because I1–9 only bound to the aggregate and the unfolding of III2 played a role in aggregation, we generated a III2 domain that was destabilized by deletion of the G strand. This mutant bound I1–9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in III2, III3, and III11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in III2 reduced the FN matrix. These results suggest that the unfolding of III2 is one of the key factors for FN aggregation and assembly. PMID:21949131

  11. A CAF40-binding motif facilitates recruitment of the CCR4-NOT complex to mRNAs targeted by Drosophila Roquin

    PubMed Central

    Sgromo, Annamaria; Raisch, Tobias; Bawankar, Praveen; Bhandari, Dipankar; Chen, Ying; Kuzuoğlu-Öztürk, Duygu; Weichenrieder, Oliver; Izaurralde, Elisa

    2017-01-01

    Human (Hs) Roquin1 and Roquin2 are RNA-binding proteins that promote mRNA target degradation through the recruitment of the CCR4-NOT deadenylase complex and are implicated in the prevention of autoimmunity. Roquin1 recruits CCR4-NOT via a C-terminal region that is not conserved in Roquin2 or in invertebrate Roquin. Here we show that Roquin2 and Drosophila melanogaster (Dm) Roquin also interact with the CCR4-NOT complex through their C-terminal regions. The C-terminal region of Dm Roquin contains multiple motifs that mediate CCR4-NOT binding. One motif binds to the CAF40 subunit of the CCR4-NOT complex. The crystal structure of the Dm Roquin CAF40-binding motif (CBM) bound to CAF40 reveals that the CBM adopts an α-helical conformation upon binding to a conserved surface of CAF40. Thus, despite the lack of sequence conservation, the C-terminal regions of Roquin proteins act as an effector domain that represses the expression of mRNA targets via recruitment of the CCR4-NOT complex. PMID:28165457

  12. Identification of the Rps28 binding motif from yeast Edc3 involved in the autoregulatory feedback loop controlling RPS28B mRNA decay.

    PubMed

    Kolesnikova, Olga; Back, Régis; Graille, Marc; Séraphin, Bertrand

    2013-11-01

    In the yeast Saccharomyces cerevisiae, the Edc3 protein was previously reported to participate in the auto-regulatory feedback loop controlling the level of the RPS28B messenger RNA (mRNA). We show here that Edc3 binds directly and tightly to the globular core of Rps28 ribosomal protein. This binding occurs through a motif that is present exclusively in Edc3 proteins from yeast belonging to the Saccharomycetaceae phylum. Functional analyses indicate that the ability of Edc3 to interact with Rps28 is not required for its general function and for its role in the regulation of the YRA1 pre-mRNA decay. In contrast, this interaction appears to be exclusively required for the auto-regulatory mechanism controlling the RPS28B mRNA decay. These observations suggest a plausible model for the evolutionary appearance of a Rps28 binding motif in Edc3.

  13. 5-Hydroxymethylcytosine in E-box motifs ACAT|GTG and ACAC|GTG increases DNA-binding of the B-HLH transcription factor TCF4.

    PubMed

    Khund-Sayeed, Syed; He, Ximiao; Holzberg, Timothy; Wang, Jun; Rajagopal, Divya; Upadhyay, Shriyash; Durell, Stewart R; Mukherjee, Sanjit; Weirauch, Matthew T; Rose, Robert; Vinson, Charles

    2016-09-12

    We evaluated DNA binding of the B-HLH family members TCF4 and USF1 using protein binding microarrays (PBMs) containing double-stranded DNA probes with cytosine on both strands or 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) on one DNA strand and cytosine on the second strand. TCF4 preferentially bound the E-box motif (CAN|NTG) with strongest binding to the 8-mer CAG|GTGGT. 5mC uniformly decreases DNA binding of both TCF4 and USF1. The bulkier 5hmC also inhibited USF1 binding to DNA. In contrast, 5hmC dramatically enhanced TCF4 binding to E-box motifs ACAT|GTG and ACAC|GTG, being better bound than any 8-mer containing cytosine. Examination of X-ray structures of the closely related TCF3 and USF1 bound to DNA suggests TCF3 can undergo a conformational shift to preferentially bind to 5hmC while the USF1 basic region is bulkier and rigid precluding a conformation shift to bind 5hmC. These results greatly expand the regulatory DNA sequence landscape bound by TCF4.

  14. Mga, a dual-specificity transcription factor that interacts with Max and contains a T-domain DNA-binding motif.

    PubMed Central

    Hurlin, P J; Steingrìmsson, E; Copeland, N G; Jenkins, N A; Eisenman, R N

    1999-01-01

    The basic-helix-loop-helix-leucine zipper (bHLHZip) proteins Myc, Mad and Mnt are part of a transcription activation/repression system involved in the regulation of cell proliferation. The function of these proteins as transcription factors is mediated by heterodimerization with the small bHLHZip protein Max, which is required for their specific DNA binding to E-box sequences. We have identified a novel Max-interacting protein, Mga, which contains a Myc-like bHLHZip motif, but otherwise shows no relationship with Myc or other Max-interacting proteins. Like Myc, Mad and Mnt proteins, Mga requires heterodimerization with Max for binding to the preferred Myc-Max-binding site CACGTG. In addition to the bHLHZip domain, Mga contains a second DNA-binding domain: the T-box or T-domain. The T-domain is a highly conserved DNA-binding motif originally defined in Brachyury and characteristic of the Tbx family of transcription factors. Mga binds the preferred Brachyury-binding sequence and represses transcription of reporter genes containing promoter-proximal Brachyury-binding sites. Surprisingly, Mga is converted to a transcription activator of both Myc-Max and Brachyury site-containing reporters in a Max-dependent manner. Our results suggest that Mga functions as a dual-specificity transcription factor that regulates the expression of both Max-network and T-box family target genes. PMID:10601024

  15. The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus.

    PubMed

    Allen, James W A; Leach, Nicholas; Ferguson, Stuart J

    2005-07-15

    c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.

  16. Presence of calcium-binding motifs in PilY1 homologs correlates with Ca-mediated twitching motility and evolutionary history across diverse bacteria.

    PubMed

    Parker, Jennifer K; Cruz, Luisa F; Evans, Michael R; De La Fuente, Leonardo

    2015-02-01

    Twitching motility, involving type IV pili, is essential for host colonization and virulence of many pathogenic bacteria. Studies of PilY1, a tip-associated type IV pili protein, indicate that PilY1 functions as a switch between pilus extension and retraction, resulting in twitching motility. Recent work detected a calcium-binding motif in PilY1 of some animal bacterial pathogens and demonstrated that binding of calcium to PilY1 with this motif regulates twitching. Though studies of PilY1 in non-animal pathogens are limited, our group demonstrated that twitching motility in the plant pathogen Xylella fastidiosa, which contains three PilY1 homologs, is increased by calcium supplementation. A study was conducted to investigate the phylogenetic relationship between multiple PilY1 homologs, the presence of calcium-binding motifs therein, and calcium-mediated twitching motility across diverse bacteria. Strains analyzed contained one to three PilY1 homologs, but phylogenetic analyses indicated that PilY1 homologs containing the calcium-binding motif Dx[DN]xDGxxD are phylogenetically divergent from other PilY1 homologs. Plant-associated bacteria included in these analyses were then examined for a calcium-mediated twitching response. Results indicate that bacteria must have at least one PilY1 homolog containing the Dx[DN]xDGxxD motif to display a calcium-mediated increase in twitching motility, which likely reflects an adaption to environmental calcium concentrations.

  17. Modification of Titanium Substrates with Chimeric Peptides Comprising Antimicrobial and Titanium-Binding Motifs Connected by Linkers To Inhibit Biofilm Formation.

    PubMed

    Liu, Zihao; Ma, Shiqing; Duan, Shun; Xuliang, Deng; Sun, Yingchun; Zhang, Xi; Xu, Xinhua; Guan, Binbin; Wang, Chao; Hu, Meilin; Qi, Xingying; Zhang, Xu; Gao, Ping

    2016-03-02

    Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p < 0.05). Concentration-dependent adsorption is consistent with a single Langmuir model, whereas time-dependent adsorption is in line with a two-domain Langmuir model. Additionally, the chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis.

  18. In silico identification of enhancers on the basis of a combination of transcription factor binding motif occurrences

    PubMed Central

    Fang, Yaping; Wang, Yunlong; Zhu, Qin; Wang, Jia; Li, Guoliang

    2016-01-01

    Enhancers interact with gene promoters and form chromatin looping structures that serve important functions in various biological processes, such as the regulation of gene transcription and cell differentiation. However, enhancers are difficult to identify because they generally do not have fixed positions or consensus sequence features, and biological experiments for enhancer identification are costly in terms of labor and expense. In this work, several models were built by using various sequence-based feature sets and their combinations for enhancer prediction. The selected features derived from a recursive feature elimination method showed that the model using a combination of 141 transcription factor binding motif occurrences from 1,422 transcription factor position weight matrices achieved a favorably high prediction accuracy superior to that of other reported methods. The models demonstrated good prediction accuracy for different enhancer datasets obtained from different cell lines/tissues. In addition, prediction accuracy was further improved by integration of chromatin state features. Our method is complementary to wet-lab experimental methods and provides an additional method to identify enhancers. PMID:27582178

  19. Identification of the Raptor-binding motif on Arabidopsis S6 kinase and its use as a TOR signaling suppressor.

    PubMed

    Son, Ora; Kim, Sunghan; Hur, Yoon-Sun; Cheon, Choong-Ill

    2016-03-25

    TOR (target of rapamycin) kinase signaling plays central role as a regulator of growth and proliferation in all eukaryotic cells and its key signaling components and effectors are also conserved in plants. Unlike the mammalian and yeast counterparts, however, we found through yeast two-hybrid analysis that multiple regions of the Arabidopsis Raptor (regulatory associated protein of TOR) are required for binding to its substrate. We also identified that a 44-amino acid region at the N-terminal end of Arabidopsis ribosomal S6 kinase 1 (AtS6K1) specifically interacted with AtRaptor1, indicating that this region may contain a functional equivalent of the TOS (TOR-Signaling) motif present in the mammalian TOR substrates. Transient over-expression of this 44-amino acid fragment in Arabidopsis protoplasts resulted in significant decrease in rDNA transcription, demonstrating a feasibility of developing a new plant-specific TOR signaling inhibitor based upon perturbation of the Raptor-substrate interaction.

  20. Novel Catalytically-Inactive PII Metalloproteinases from a Viperid Snake Venom with Substitutions in the Canonical Zinc-Binding Motif

    PubMed Central

    Camacho, Erika; Sanz, Libia; Escalante, Teresa; Pérez, Alicia; Villalta, Fabián; Lomonte, Bruno; Neves-Ferreira, Ana Gisele C.; Feoli, Andrés; Calvete, Juan J.; Gutiérrez, José María; Rucavado, Alexandra

    2016-01-01

    Snake venom metalloproteinases (SVMPs) play key biological roles in prey immobilization and digestion. The majority of these activities depend on the hydrolysis of relevant protein substrates in the tissues. Hereby, we describe several isoforms and a cDNA clone sequence, corresponding to PII SVMP homologues from the venom of the Central American pit viper Bothriechis lateralis, which have modifications in the residues of the canonical sequence of the zinc-binding motif HEXXHXXGXXH. As a consequence, the proteolytic activity of the isolated proteins was undetectable when tested on azocasein and gelatin. These PII isoforms comprise metalloproteinase and disintegrin domains in the mature protein, thus belonging to the subclass PIIb of SVMPs. PII SVMP homologues were devoid of hemorrhagic and in vitro coagulant activities, effects attributed to the enzymatic activity of SVMPs, but induced a mild edema. One of the isoforms presents the characteristic RGD sequence in the disintegrin domain and inhibits ADP- and collagen-induced platelet aggregation. Catalytically-inactive SVMP homologues may have been hitherto missed in the characterization of snake venoms. The presence of such enzymatically-inactive homologues in snake venoms and their possible toxic and adaptive roles deserve further investigation. PMID:27754342

  1. New molecular motif for recognizing sialic acid using emissive lanthanide-macrocyclic polyazacarboxylate complexes: deprotonation of a coordinated water molecule controls specific binding.

    PubMed

    Ouchi, Kazuki; Saito, Shingo; Shibukawa, Masami

    2013-06-03

    A new molecular motif--lanthanide-macrocyclic polyazacarboxylate hexadentate complexes, Ln(3+)-ABNOTA--was found to specifically bind to sialic acid with strong emission enhancement and high affinity. The selectivity toward sialic acid over other monosaccharides was one of the highest among artificial receptors. Also, the novel binding mechanism was investigated in detail; binding selectivity is controlled by interactions between sialic acid and both the central metal and a hydroxyl group produced by deprotonation of a coordinated water molecule in the Ln(3+) complex.

  2. A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells.

    PubMed Central

    Moyano, José V; Maqueda, Alfredo; Albar, Juan P; Garcia-Pardo, Angeles

    2003-01-01

    Cell adhesion to fibronectin results in formation of actin stress fibres and focal adhesions. In fibroblasts, this response requires two co-operative signals provided by interactions of the RGD sequence with alpha5beta1 integrin and the heparin-binding domain II (Hep II) domain with syndecan-4. Within Hep II, this activity was mapped to repeat III13 and to the peptide FN-C/H-V(WQPPRARITGY, repeat III14). We previously described that the synthetic heparin-binding peptide/III5 (HBP/III5) (WTPPRAQITGYRLTVGLTRR, repeat III5) binds heparin and mediates cell adhesion via chondroitin sulphate proteoglycans. We have now studied whether HBP/III5 co-operates with alpha5beta1 and drives a full cytoskeletal response in melanoma cells. SKMEL-178 cells attached and spread on the RGD-containing FNIII7-FNIII10 (FNIII7-10) fragment, but did not form stress fibres or focal adhesions. Co-immobilization of HBP/III5 with FNIII7-10 or adding soluble HBP/III5 to cells prespread on FNIII7-10, effectively induced these structures. Cell transfection with dominant-negative N19RhoA, a member of the small GTPase family, abolished the HBP/III5 effect. Both chondroitinase and heparitinase diminished focal adhesions, indicating that both types of proteoglycans bound HBP/III5 in melanoma cells. We have mapped the active sequence of HBP/III5 to YRLTVGLTRR, which is a novel sequence in fibronectin with focal-adhesion-promoting activity. The last two arginine (R) residues of this sequence are required for activity, since their replacement by alanine completely abrogated the HBP/III5 cytoskeletal effect. Moreover, this sequence is also active in the context of large fibronectin fragments. Our results establish that the Hep III region provides co-operative signals to alpha5beta1 for the progression of the cytoskeletal response and that these include activation of RhoA. PMID:12519080

  3. Flotillins bind to the dileucine sorting motif of β-site amyloid precursor protein-cleaving enzyme 1 and influence its endosomal sorting.

    PubMed

    John, Bincy A; Meister, Melanie; Banning, Antje; Tikkanen, Ritva

    2014-04-01

    The β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a protease that participates in the amyloidogenic cleavage of the Alzheimer amyloid precursor protein. Trafficking of BACE1 has been shown to be largely mediated by an acidic cluster dileucine motif in its cytoplasmic tail. This sorting signal functions both in endocytosis and endosomal sorting/recycling of BACE1 by providing a binding site for various sorting factors, such as the Golgi-localizing γ-ear containing ADP ribosylation factor binding (GGA) proteins that mediate BACE1 sorting within endosomes. Because flotillin-1 has been suggested to bind to BACE1 cytoplasmic tail, we analyzed the role of flotillins in BACE1 sorting. We show that flotillin-1 directly binds to the dileucine motif in the cytoplasmic tail of BACE1, whereas flotillin-2 binding is mainly mediated by its interaction with flotillin-1. Depletion of flotillins results in altered subcellular localization of BACE1 in endosomes and stabilization of BACE1 protein. Furthermore, amyloidogenic processing of Alzheimer amyloid precursor protein is increased. Flotillins compete with GGA proteins for binding to the dileucine motif in the BACE1 tail, suggesting that they play an important role in endosomal sorting of BACE1. The present study shows for the first time that flotillins are involved in endosomal sorting of BACE1. Because the endosomal localization of BACE1 affects its function as the β-secretase by increasing amyloidogenic processing of the amyloid precursor protein, flotillins may play a novel role in Alzheimer's disease. The present study is the first to show that flotillins bind to a canonical sorting signal and influence the binding of endosomal sorting factors onto cargo tails.

  4. Identification of Streptococcus mutans PAc peptide motif binding with human MHC class II molecules (DRB1*0802, *1101, *1401 and *1405).

    PubMed Central

    Senpuku, H; Yanagi, K; Nisizawa, T

    1998-01-01

    A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population. PMID:9824493

  5. Identification of Streptococcus mutans PAc peptide motif binding with human MHC class II molecules (DRB1*0802, *1101, *1401 and *1405).

    PubMed

    Senpuku, H; Yanagi, K; Nisizawa, T

    1998-11-01

    A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population.

  6. Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.

    PubMed

    Turner, C E; Brown, M C; Perrotta, J A; Riedy, M C; Nikolopoulos, S N; McDonald, A R; Bagrodia, S; Thomas, S; Leventhal, P S

    1999-05-17

    Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

  7. Genetics Home Reference: fibronectin glomerulopathy

    MedlinePlus

    ... tiny blood vessels in the kidneys that filter waste products from blood . The waste products are then released in urine. The fibronectin- ... the body attempts to filter it out as waste . Even though there is an abundance of fibronectin- ...

  8. Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.

    PubMed

    Nagashima, Shunta; Maruyama, Junichi; Kawano, Shodai; Iwasa, Hiroaki; Nakagawa, Kentaro; Ishigami-Yuasa, Mari; Kagechika, Hiroyuki; Nishina, Hiroshi; Hata, Yutaka

    2016-06-01

    Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial-mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP-fused TAZ (GFP-TAZ), monitored the subcellular localization of GFP-TAZ, and selected 33 compounds that shifted GFP-TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ-mediated enhancement of TEAD-responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP-TAZ-based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds.

  9. Role of NH{sub 2}-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: Common features in eukaryotic organisms

    SciTech Connect

    Lee, Asaka; Asahina, Kota; Okamoto, Takumi; Kawaguchi, Kosuke; Kostsin, Dzmitry G.; Kashiwayama, Yoshinori; Takanashi, Kojiro; Yazaki, Kazufumi; Imanaka, Tsuneo; Morita, Masashi

    2014-10-24

    Highlights: • ABCD proteins classifies based on with or without NH{sub 2}-terminal hydrophobic segment. • The ABCD proteins with the segment are targeted peroxisomes. • The ABCD proteins without the segment are targeted to the endoplasmic reticulum. • The role of the segment in organelle targeting is conserved in eukaryotic organisms. - Abstract: In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1–3 possesses the NH{sub 2}-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH{sub 2}-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH{sub 2}-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH{sub 2}-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH{sub 2}-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH{sub 2}-terminal H0 motif in organelle targeting is widely conserved in living organisms.

  10. Developmentally Regulated RNA-binding Protein 1 (Drb1)/RNA-binding Motif Protein 45 (RBM45), a Nuclear-Cytoplasmic Trafficking Protein, Forms TAR DNA-binding Protein 43 (TDP-43)-mediated Cytoplasmic Aggregates.

    PubMed

    Mashiko, Takafumi; Sakashita, Eiji; Kasashima, Katsumi; Tominaga, Kaoru; Kuroiwa, Kenji; Nozaki, Yasuyuki; Matsuura, Tohru; Hamamoto, Toshiro; Endo, Hitoshi

    2016-07-15

    Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drb1)/RNA-binding motif protein 45 is an RNA-binding protein that was recently described as a component in ALS- and FTLD-related inclusion bodies. However, the molecular mechanism underlying cytoplasmic Drb1 aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drb1 co-localizes with cytoplasmic aggregates mediated by TAR DNA-binding protein 43, a major component of ALS and FTLD-related inclusion bodies. We also defined the domains involved in the subcellular localization of Drb1 to clarify the role of Drb1 in the formation of cytoplasmic aggregates in ALS and FTLD. Drb1 predominantly localized in the nucleus via a classical nuclear localization signal in its carboxyl terminus and is a shuttling protein between the nucleus and cytoplasm. Furthermore, we identify a double leucine motif serving as a nuclear export signal. The Drb1 mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb1-induced cytoplasmic aggregates not only recruit TAR DNA-binding protein 43 but also decrease the mitochondrial membrane potential. Taken together, these results indicate that perturbation of Drb1 nuclear-cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells.

  11. Biophysical analysis of binding of WW domains of the YAP2 transcriptional regulator to PPXY motifs within WBP1 and WBP2 adaptors.

    PubMed

    McDonald, Caleb B; McIntosh, Samantha K N; Mikles, David C; Bhat, Vikas; Deegan, Brian J; Seldeen, Kenneth L; Saeed, Ali M; Buffa, Laura; Sudol, Marius; Nawaz, Zafar; Farooq, Amjad

    2011-11-08

    The YAP2 transcriptional regulator mediates a plethora of cellular functions, including the newly discovered Hippo tumor suppressor pathway, by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using isothermal titration calorimery and circular dichroism in combination with molecular modeling and molecular dynamics, we provide evidence that the WW1 and WW2 domains of YAP2 recognize various PPXY motifs within WBP1 and WBP2 in a highly promiscuous and subtle manner. Thus, although both WW domains strictly require the integrity of the consensus PPXY sequence, nonconsensus residues within and flanking this motif are not critical for high-affinity binding, implying that they most likely play a role in stabilizing the polyproline type II helical conformation of the PPXY ligands. Of particular interest is the observation that both WW domains bind to a PPXYXG motif with highest affinity, implicating a preference for a nonbulky and flexible glycine one residue to the C-terminal side of the consensus tyrosine. Importantly, a large set of residues within both WW domains and the PPXY motifs appear to undergo rapid fluctuations on a nanosecond time scale, suggesting that WW-ligand interactions are highly dynamic and that such conformational entropy may be an integral part of the reversible and temporal nature of cellular signaling cascades. Collectively, our study sheds light on the molecular determinants of a key WW-ligand interaction pertinent to cellular functions in health and disease.

  12. Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets.

    PubMed

    Nelson, Christopher S; Fuller, Chris K; Fordyce, Polly M; Greninger, Alexander L; Li, Hao; DeRisi, Joseph L

    2013-07-01

    The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein's DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2's-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved.

  13. Specificity in substrate binding by protein folding catalysts: tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp.

    PubMed Central

    Ruddock, L. W.; Freedman, R. B.; Klappa, P.

    2000-01-01

    Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding. PMID:10794419

  14. The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes.

    PubMed

    Benito-Jardón, Maria; Klapproth, Sarah; Gimeno-LLuch, Irene; Petzold, Tobias; Bharadwaj, Mitasha; Müller, Daniel J; Zuchtriegel, Gabriele; Reichel, Christoph A; Costell, Mercedes

    2017-01-16

    Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5β1, αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1(syn/syn)) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of β3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5β1 with the RGD, but essential to re-enforce the binding of α5β1/αIIbβ3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvβ3 integrin levels decrease below a critical level.

  15. The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes

    PubMed Central

    Benito-Jardón, Maria; Klapproth, Sarah; Gimeno-LLuch, Irene; Petzold, Tobias; Bharadwaj, Mitasha; Müller, Daniel J; Zuchtriegel, Gabriele; Reichel, Christoph A; Costell, Mercedes

    2017-01-01

    Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5β1, αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of β3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5β1 with the RGD, but essential to re-enforce the binding of α5β1/αIIbβ3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvβ3 integrin levels decrease below a critical level. DOI: http://dx.doi.org/10.7554/eLife.22264.001 PMID:28092265

  16. The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats

    PubMed Central

    Bowman, Andrew; Lercher, Lukas; Singh, Hari R.; Zinne, Daria; Timinszky, Gyula; Carlomagno, Teresa; Ladurner, Andreas G.

    2016-01-01

    Eukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from 1H-15N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell. PMID:26673727

  17. The MotA transcription factor from bacteriophage T4 contains a novel DNA-binding domain : the 'double wing' motif.

    SciTech Connect

    Li, N.; Sickmier, E. A.; Zhang, R.; Joachimiak, A.; White, S. W.; Biosciences Division; St. Jude Children's Research Hospital; Univ. of Tennessee Health Science Center; Corixa Inc.

    2002-01-01

    MotA is a transcription factor from bacteriophage T4 that helps adapt the host Escherichia coli transcription apparatus to T4 middle promoters. We have determined the crystal structure of the C-terminal DNA-binding domain of MotA (MotCF) to 1.6 A resolution using multiwavelength, anomalous diffraction methods. The structure reveals a novel DNA-binding alpha/beta motif that contains an exposed beta-sheet surface that mediates interactions with the DNA. Independent biochemical experiments have shown that MotCF binds to one surface of a single turn of DNA through interactions in adjacent major and minor grooves. We present a model of the interaction in which beta-ribbons at opposite corners of the six-stranded beta-sheet penetrate the DNA grooves, and call the motif a 'double wing' to emphasize similarities to the 'winged-helix' motif. The model is consistent with data on how MotA functions at middle promoters, and provides an explanation for why MotA can form non-specific multimers on DNA.

  18. The dual role of LSD1 and HDAC3 in STAT5-dependent transcription is determined by protein interactions, binding affinities, motifs and genomic positions

    PubMed Central

    Nanou, Aikaterini; Toumpeki, Chrisavgi; Lavigne, Matthieu D.; Lazou, Vassiliki; Demmers, Jeroen; Paparountas, Triantafillos; Thanos, Dimitris; Katsantoni, Eleni

    2017-01-01

    STAT5 interacts with other factors to control transcription, and the mechanism of regulation is of interest as constitutive active STAT5 has been reported in malignancies. Here, LSD1 and HDAC3 were identified as novel STAT5a interacting partners in pro-B cells. Characterization of STAT5a, LSD1 and HDAC3 target genes by ChIP-seq and RNA-seq revealed gene subsets regulated by independent or combined action of the factors and LSD1/HDAC3 to play dual role in their activation or repression. Genes bound by STAT5a alone or in combination with weakly associated LSD1 or HDAC3 were enriched for the canonical STAT5a GAS motif, and such binding induced activation or repression. Strong STAT5 binding was seen more frequently in intergenic regions, which might function as distal enhancer elements. Groups of genes bound weaker by STAT5a and stronger by LSD1/HDAC3 showed an absence of the GAS motif, and were differentially regulated based on their genomic binding localization and binding affinities. These genes exhibited increased binding frequency in promoters, and in conjunction with the absence of GAS sites, the data indicate a requirement for stabilization by additional factors, which might recruit LSD1/HDAC3. Our study describes an interaction network of STAT5a/LSD1/HDAC3 and a dual function of LSD1/HDAC3 on STAT5-dependent transcription, defined by protein–protein interactions, genomic binding localization/affinity and motifs. PMID:27651463

  19. Identification of second arginine-glycine-aspartic acid motif of ovine vitronectin as the complement C9 binding site and its implication in bacterial infection.

    PubMed

    T, Prasada Rao; T, Lakshmi Prasanth; R, Parvathy; S, Murugavel; Devi, Karuna; Joshi, Paritosh

    2017-02-02

    Vitronectin (Vn), a multifunctional protein of blood and extracellular matrix interacts with complement C9. This interaction may modulate innate immunity. Details of Vn-C9 interaction are limited. An assessment of Vn-C9 interaction was made employing goat homologous system. Vn binding to C9 was observed in three different assays. Using recombinant fragments, the C9 binding was mapped to the N-terminus of Vn. Site directed mutagenesis was performed to alter the second RGD sequence (RGD-2) of Vn. Change of R to G or D to A in RGD-2 caused significant decrease in Vn binding to C9 whereas change of R to G in the first RGD motif (RGD-1) had no effect on Vn binding to C9. These results imply that the RGD-2 of goat Vn is involved in C9 binding. In competitive binding assay, the presence of soluble RGD peptide inhibited Vn binding to C9 whereas heparin had no effect. Vn binding to C9 in terms of bacterial pathogenesis was also evaluated. Serum dependent inhibition of E. coli growth was significantly reverted when Vn or its N-fragment were included in the assay. The C-fragment, which did not support C9 binding, also partly nullified serum dependent inhibition of bacterial growth probably through other serum component(s).

  20. Ehrlichia chaffeensis TRP120 binds a G+C-rich motif in host cell DNA and exhibits eukaryotic transcriptional activator function.

    PubMed

    Zhu, Bing; Kuriakose, Jeeba A; Luo, Tian; Ballesteros, Efren; Gupta, Sharu; Fofanov, Yuriy; McBride, Jere W

    2011-11-01

    Ehrlichia chaffeensis is an obligately intracellular bacterium that modulates host cell gene transcription in the mononuclear phagocyte, but the host gene targets and mechanisms involved in transcriptional modulation are not well-defined. In this study, we identified a novel tandem repeat DNA-binding domain in the E. chaffeensis 120-kDa tandem repeat protein (TRP120) that directly binds host cell DNA. TRP120 was observed by immunofluorescent microscopy in the nucleus of E. chaffeensis-infected host cells and was detected in nuclear extracts by Western immunoblotting with TRP120-specific antibody. The TRP120 binding sites and associated host cell target genes were identified using high-throughput deep sequencing (Illumina) of immunoprecipitated DNA (chromatin immunoprecipitation and high-throughput DNA sequencing). Multiple em motif elicitation (MEME) analysis of the most highly enriched TRP120-bound sequences revealed a G+C-rich DNA motif, and recombinant TRP120 specifically bound synthetic oligonucleotides containing the motif. TRP120 target gene binding sites were mapped most frequently to intersecting regions (intron/exon; 49%) but were also identified in upstream regulatory regions (25%) and downstream locations (26%). Genes targeted by TRP120 were most frequently associated with transcriptional regulation, signal transduction, and apoptosis. TRP120 targeted inflammatory chemokine genes, CCL2, CCL20, and CXCL11, which were strongly upregulated during E. chaffeensis infection and were also upregulated by direct transfection with recombinant TRP120. This study reveals that TRP120 is a novel DNA-binding protein that is involved in a host gene transcriptional regulation strategy.

  1. Characterization of the Canine MHC Class I DLA-88*50101 Peptide Binding Motif as a Prerequisite for Canine T Cell Immunotherapy

    PubMed Central

    Barth, Sharon M.; Schreitmüller, Christian M.; Proehl, Franziska; Oehl, Kathrin; Lumpp, Leonie M.; Kowalewski, Daniel J.; Di Marco, Moreno; Sturm, Theo; Backert, Linus; Schuster, Heiko; Stevanović, Stefan; Rammensee, Hans-Georg; Planz, Oliver

    2016-01-01

    There are limitations in pre-clinical settings using mice as a basis for clinical development in humans. In cancer, similarities exist between humans and dogs; thus, the dog patient can be a link in the transition from laboratory research on mouse models to clinical trials in humans. Knowledge of the peptides presented on MHC molecules is fundamental for the development of highly specific T cell-based immunotherapies. This information is available for human MHC molecules but is absent for the canine MHC. In the present study, we characterized the binding motif of dog leukocyte antigen (DLA) class I allele DLA-88*50101, using human C1R and K562 transfected cells expressing the DLA-88*50101 heavy chain. MHC class I immunoaffinity-purification revealed 3720 DLA-88*50101 derived peptides, which enabled the determination of major anchor positions. The characterized binding motif of DLA-88*50101 was similar to HLA-A*02:01. Peptide binding analyses on HLA-A*02:01 and DLA-88*50101 via flow cytometry showed weak binding of DLA-88*50101 derived peptides to HLA-A*02:01, and vice versa. Our results present for the first time a detailed peptide binding motif of the canine MHC class I allelic product DLA-88*50101. These data support the goal of establishing dogs as a suitable animal model for the evaluation and development of T cell-based cancer immunotherapies, benefiting both dog and human patients. PMID:27893789

  2. PDZ Domain Dependent Regulation of NHE3 Occurs by Both Internal Class II and C-terminal Class I PDZ Binding Motifs.

    PubMed

    Cha, Boyoung; Yang, Jianbo; Singh, Varsha; Zachos, Nicholas C; Sarker, Rafiquel I; Chen, Tian-E; Chakraborty, Molee; Tse, Chung-Ming; Donowitz, Mark

    2017-03-10

    NHE3 directly binds NHERF family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (aa. 586-660) of the NHE3 C-terminus and to the NHE3 C-terminal four amino acids. The internal NHERF binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ binding motifs (PBM). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing: 1) Interaction between these binding sites since mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression were reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP and Ca2+-mediated inhibition of NHE3 were impaired both in the internal and in the C-terminal PBM mutations. 4) A significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) Some difference in NHE3 associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II and C-terminal Class I PBM, which interact. The former appears to determine NHE3 stability of a pool in the PM and the letter determines total expression and percent PM expression.

  3. Exploratory Study on the RNA-Binding Structural Motifs by Library Screening Targeting pre-miRNA-29 a.

    PubMed

    Fukuzumi, Takeo; Murata, Asako; Aikawa, Haruo; Harada, Yasue; Nakatani, Kazuhiko

    2015-11-16

    The metabolic stream of microRNA (miRNA) production, the so-called maturation process of miRNAs, became one of important metabolic paths for drug-targeting to modulate the expression of genes related to a number of diseases. We carried out discovery studies on small molecules binding to the precursor of miR-29a (pre-miR-29a) from a chemical library containing 41,119 compounds (AQ library) by the fluorescent indicator displacement (FID) assay using the xanthone derivative X2SdiMe as a fluorescent indicator. The FID assay provided 1075 compounds, which showed an increase of fluorescence. These compounds were subsequently submitted to a binding analysis in a surface plasmon resonance (SPR) assay on a pre-miR-29a immobilized surface. 21 hit compounds were identified with a good reproducibility in the binding. These compounds have not been reported to bind to RNA until now and can be classified into two groups on the basis of the kinetics in the binding. To gain more information on the motif structures that could be necessary for the binding to pre-miR-29a, 19 substructures were selected from the hit compounds. The substructure library (SS library) which consisted of 362 compounds was prepared from the AQ library. An SPR assay of the SS library on pre-miR-29a-immobilized surface suggested that five substructures could potentially be important structural motifs to bind to pre-miR-29a. These studies demonstrate that the combination of FID-based screening of chemical library and subsequent SPR assay would be one way for obtaining practical solutions for the discovery of molecules which bind to the target pre-miRNAs.

  4. Using Weeder, Pscan, and PscanChIP for the Discovery of Enriched Transcription Factor Binding Site Motifs in Nucleotide Sequences.

    PubMed

    Zambelli, Federico; Pesole, Graziano; Pavesi, Giulio

    2014-09-08

    One of the greatest challenges facing modern molecular biology is understanding the complex mechanisms regulating gene expression. A fundamental step in this process requires the characterization of sequence motifs involved in the regulation of gene expression at transcriptional and post-transcriptional levels. In particular, transcription is modulated by the interaction of transcription factors (TFs) with their corresponding binding sites. Weeder, Pscan, and PscanChIP are software tools freely available for noncommercial users as a stand-alone or Web-based applications for the automatic discovery of conserved motifs in a set of DNA sequences likely to be bound by the same TFs. Input for the tools can be promoter sequences from co-expressed or co-regulated genes (for which Weeder and Pscan are suitable), or regions identified through genome wide ChIP-seq or similar experiments (Weeder and PscanChIP). The motifs are either found by a de novo approach (Weeder) or by using descriptors of the binding specificity of TFs (Pscan and PscanChIP).

  5. Downstream signaling molecules bind to different phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) peptides of the high affinity IgE receptor.

    PubMed

    Kimura, T; Kihara, H; Bhattacharyya, S; Sakamoto, H; Appella, E; Siraganian, R P

    1996-11-01

    The cytoplasmic tails of both the beta and gamma subunits of the high affinity IgE receptor (FcepsilonRI) contain a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). This motif plays a critical role in receptor-mediated signal transduction. Synthetic peptides based on the ITAM sequences of the beta and gamma subunits of FcepsilonRI were used to investigate which proteins associate with these motifs. Tyrosine-phosphorylated beta and gamma ITAM peptides immobilized on beads precipitated Syk, Lyn, Shc, Grb2, and phospholipase C-gamma1 from lysates of rat basophilic leukemia RBL-2H3 cells. Syk was precipitated predominantly by the tyrosine-diphosphorylated gamma ITAM peptide, but much less by the diphosphorylated beta ITAM peptide or by the monophosphorylated peptides. Phospholipase C-gamma1, Shc, and Grb2 were precipitated only by the diphosphorylated beta ITAM peptide. Non-phosphorylated ITAM peptides did not precipitate these proteins. In membrane binding assays, fusion proteins containing the Src homology 2 domains of phospholipase C-gamma1, Shc, Syk, and Lyn directly bound the tyrosine-phosphorylated ITAM peptides. Although the ITAM sequences of the beta and gamma subunits of FcepsilonRI are similar, once they are tyrosine-phosphorylated they preferentially bind different downstream signaling molecules. Tyrosine phosphorylation of the ITAM of the gamma subunit recruits and activates Syk, whereas the beta subunit may be important for the Ras signaling pathway.

  6. Nonclathrin coat protein gamma, a subunit of coatomer, binds to the cytoplasmic dilysine motif of membrane proteins of the early secretory pathway.

    PubMed Central

    Harter, C; Pavel, J; Coccia, F; Draken, E; Wegehingel, S; Tschochner, H; Wieland, F

    1996-01-01

    Coatomer, a cytosolic heterooligomeric protein complex that consists of seven subunits [alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP (nonclathrin coat protein)], has been shown to interact with dilysine motifs typically found in the cytoplasmic domains of various endoplasmic-reticulum-resident membrane proteins [Cosson, P. & Letourneur, F. (1994) Science 263, 1629-1631]. We have used a photo-cross-linking approach to identify the site of coatomer that is involved in binding to the dilysine motifs. An octapeptide corresponding to the C-terminal tail of Wbp1p, a component of the yeast N-oligosaccharyltransferase complex, has been synthesized with a photoreactive phenylalanine at position -5 and was radioactively labeled with [125I]iodine at a tyrosine residue introduced at the N terminus of the peptide. Photolysis of isolated coatomer in the presence of this peptide and immunoprecipitation of coatomer from photo-cross-linked cell lysates reveal that gamma-COP is the predominantly labeled protein. From these results, we conclude that coatomer is able to bind to the cytoplasmic dilysine motifs of membrane proteins of the early secretory pathway via its gamma-COP subunit, whose complete cDNA-derived amino acid sequence is also presented. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8700856

  7. ATG4B contains a C-terminal LIR motif important for binding and efficient cleavage of mammalian orthologs of yeast Atg8.

    PubMed

    Skytte Rasmussen, Mads; Mouilleron, Stéphane; Kumar Shrestha, Birendra; Wirth, Martina; Lee, Rebecca; Bowitz Larsen, Kenneth; Abudu Princely, Yakubu; O'Reilly, Nicola; Sjøttem, Eva; Tooze, Sharon A; Lamark, Trond; Johansen, Terje

    2017-02-15

    The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.

  8. Different motif requirements for the localization zipcode element of β-actin mRNA binding by HuD and ZBP1

    PubMed Central

    Kim, Hak Hee; Lee, Seung Joon; Gardiner, Amy S.; Perrone-Bizzozero, Nora I.; Yoo, Soonmoon

    2015-01-01

    Interactions of RNA-binding proteins (RBPs) with their target transcripts are essential for regulating gene expression at the posttranscriptional level including mRNA export/localization, stability, and translation. ZBP1 and HuD are RBPs that play pivotal roles in mRNA transport and local translational control in neuronal processes. While HuD possesses three RNA recognition motifs (RRMs), ZBP1 contains two RRMs and four K homology (KH) domains that either increase target specificity or provide a multi-target binding capability. Here we used isolated cis-element sequences of the target mRNA to examine directly protein-RNA interactions in cell-free systems. We found that both ZBP1 and HuD bind the zipcode element in rat β-actin mRNA's 3′ UTR. Differences between HuD and ZBP1 were observed in their binding preference to the element. HuD showed a binding preference for U-rich sequence. In contrast, ZBP1 binding to the zipcode RNA depended more on the structural level, as it required the proper spatial organization of a stem-loop that is mainly determined by the U-rich element juxtaposed to the 3′ end of a 5′-ACACCC-3′ motif. On the basis of this work, we propose that ZBP1 and HuD bind to overlapping sites in the β-actin zipcode, but they recognize different features of this target sequence. PMID:26152301

  9. Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ.

    PubMed

    Baraquet, Claudine; Harwood, Caroline S

    2013-11-12

    The transcription factor FleQ is a bacterial AAA+ ATPase enhancer-binding protein that is the master activator of flagella gene expression in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Homologs of FleQ are present in all Pseudomonas species and in many polarly flagellated gamma proteobacteria. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls the transition between planktonic and biofilm modes of growth in bacteria in response to diverse environmental signals. C-di-GMP binds to FleQ to dampen its activity, causing down-regulation of flagella gene expression. This action is potentiated in the simultaneous presence of another protein, FleN. We explored the effect of c-di-GMP and FleN on the ATPase activity of FleQ and found that a relatively low concentration of c-di-GMP competitively inhibited FleQ ATPase activity, suggesting that c-di-GMP competes with ATP for binding to the Walker A motif of FleQ. Confirming this, a FleQ Walker A motif mutant failed to bind c-di-GMP. FleN, whose gene is regulated by FleQ, also inhibited FleQ ATPase activity, and FleQ ATPase activity was much more inhibited by c-di-GMP in the presence of FleN than in its absence. These results indicate that FleN and c-di-GMP cooperate to inhibit FleQ activity and, by extension, flagella synthesis in P. aeruginosa. The Walker A motif of FleQ is perfectly conserved, opening up the possibility that other AAA+ ATPases may respond to c-di-GMP.

  10. Inhibition of NF-kappaB by ZAS3, a zinc-finger protein that also binds to the kappaB motif.

    PubMed

    Hong, Joung-Woo; Allen, Carl E; Wu, Lai-Chu

    2003-10-14

    The ZAS proteins are large zinc-finger transcriptional proteins implicated in growth, signal transduction, and lymphoid development. Recombinant ZAS fusion proteins containing one of the two DNA-binding domains have been shown to bind specifically to the kappaB motif, but the endogenous ZAS proteins or their physiological functions are largely unknown. The kappaB motif, GGGACTTTCC, is a gene regulatory element found in promoters and enhancers of genes involved in immunity, inflammation, and growth. The Rel family of NF-kappaB, predominantly p65.p50 and p50.p50, are transcription factors well known for inducing gene expression by means of interaction with the kappaB motif during acute-phase responses. A functional link between ZAS and NF-kappaB, two distinct families of kappaB-binding proteins, stems from our previous in vitro studies that show that a representative member, ZAS3, associates with TRAF2, an adaptor molecule in tumor necrosis factor signaling, to inhibit NF-kappaB activation. Biochemical and genetic evidence presented herein shows that ZAS3 encodes major kappaB-binding proteins in B lymphocytes, and that NF-kappaB is constitutively activated in ZAS3-deficient B cells. The data suggest that ZAS3 plays crucial functions in maintaining cellular homeostasis, at least in part by inhibiting NF-kappaB by means of three mechanisms: inhibition of nuclear translocation of p65, competition for kappaB gene regulatory elements, and repression of target gene transcription.

  11. Unusual conformation of the SxN motif in the crystal structure of penicillin-binding protein A from Mycobacterium tuberculosis.

    SciTech Connect

    Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

    2010-07-19

    PBPA from Mycobacterium tuberculosis is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in M. tuberculosis, but is important for proper cell division in Mycobacterium smegmatis. We have determined the crystal structure of PBPA at 2.05 {angstrom} resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain, and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase fold and contains the three conserved active-site motifs characterisitic of penicillin-interacting enzymes. While the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulfide bridge between Cys282 (the 'x' of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between {beta}5 and {alpha}11, which restricts the space available in the active site of PBPA and suggests that conformational changes would be required to accommodate peptide substrate or {beta}-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.

  12. Fibronectin deposition in delayed-type hypersensitivity. Reactions of normals and a patient with afibrinogenemia.

    PubMed Central

    Clark, R A; Horsburgh, C R; Hoffman, A A; Dvorak, H F; Mosesson, M W; Colvin, R B

    1984-01-01

    During development of delayed hypersensitivity (DH) skin reactions, fibronectin accumulates in two distinct sites: (a) the dermal interstitium in a pattern similar to fibrin and with a time course similar to that of fibrin deposition and mononuclear cell infiltration, and (b) blood vessel walls in a pattern suggestive of basement membrane staining and with a time course similar to that of endothelial cell proliferation. In vitro fibronectin can bind to monocytes or endothelial cells and simultaneously bind to fibrin or collagen matrices; by such interaction in vivo it may affect cell migration or proliferation. Thus, fibronectin deposition in DH reactions may facilitate cell-matrix interactions; however, the possibility exists that extravascular fibronectin accumulation may be only secondary to interstitial fibrin clot formation, and that blood vessel-associated fibronectin may be only a function of adsorption onto basement membrane (type IV) collagen. To address these possibilities, we investigated the association of fibronectin with fibrin, type IV collagen, and mononuclear cell infiltrates in DH reactions. Skin sites of DH reactions in normal volunteers were biopsied at 24, 48, and 72 h after intradermal challenge and examined by immunofluorescence technique. At all time points most of the interstitial fibronectin coincided with fibrin; however, some interstitial fibronectin was coincident with mononuclear cells positive for HLA-DR or monocyte-specific antigen. The coincidence of fibronectin with mononuclear cells was more apparent in a 48-h DH reaction from a patient with congenital afibrinogenemia. Vessel wall fibronectin was increased by 48 h after challenge and appeared as a fine linear band on the luminal side of a much thicker band of type IV collagen. Thus, the coincidence of extravascular fibronectin with mononuclear cells, its appearance without fibrin in the site from a patient with afibrinogenemia, and incomplete correspondence of vessel wall

  13. Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco.

    PubMed

    Felix, Georg; Boller, Thomas

    2003-02-21

    To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs). Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco. Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations. Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes. Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells. Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs. Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity. Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors. Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested. Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.

  14. Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing*

    PubMed Central

    Pérez-Cañamás, Miryam; Hernández, Carmen

    2015-01-01

    Viruses express viral suppressors of RNA silencing (VSRs) to counteract RNA silencing-based host defenses. Although virtually all stages of the antiviral silencing pathway can be inhibited by VSRs, small RNAs (sRNAs) and Argonaute (AGO) proteins seem to be the most frequent targets. Recently, GW/WG motifs of some VSRs have been proposed to dictate their suppressor function by mediating interaction with AGO(s). Here we have studied the VSR encoded by Pelargonium line pattern virus (family Tombusviridae). The results show that p37, the viral coat protein, blocks RNA silencing. Site-directed mutagenesis of some p37 sequence traits, including a conserved GW motif, allowed generation of suppressor-competent and -incompetent molecules and uncoupling of the VSR and particle assembly capacities. The engineered mutants were used to assess the importance of p37 functions for viral infection and the relative contribution of diverse molecular interactions to suppressor activity. Two main conclusions can be drawn: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic Pelargonium line pattern virus infection, and (ii) the suppressor activity of p37 relies on the ability to bind sRNAs rather than on interaction with AGOs. The data also caution against potential misinterpretations of results due to overlap of sequence signals related to distinct protein properties. This is well illustrated by mutation of the GW motif in p37 that concurrently affects nucleolar localization, efficient interaction with AGO1, and sRNA binding capability. These concomitant effects could have been overlooked in other GW motif-containing suppressors, as we exemplify with the orthologous p38 of turnip crinkle virus. PMID:25505185

  15. Crystal structure of the N-terminal region of human Ash2L shows a winged-helix motif involved in DNA binding

    SciTech Connect

    Chen, Yong; Wan, Bingbing; Wang, Kevin C.; Cao, Fang; Yang, Yuting; Protacio, Angeline; Dou, Yali; Chang, Howard Y.; Lei, Ming

    2011-09-06

    Ash2L is a core component of the MLL family histone methyltransferases and has an important role in regulating the methylation of histone H3 on lysine 4. Here, we report the crystal structure of the N-terminal domain of Ash2L and reveal a new function of Ash2L. The structure shows that Ash2L contains an atypical PHD finger that does not have histone tail-binding activity. Unexpectedly, the structure shows a previously unrecognized winged-helix motif that directly binds to DNA. The DNA-binding-deficient mutants of Ash2L reduced Ash2L localization to the HOX locus. Strikingly, a single mutation in Ash2L{sub WH} (K131A) breaks the chromatin domain boundary, suggesting that Ash2L also has a role in chromosome demarcation.

  16. Gas-phase complexes of Ni2+ and Ca2+ with deprotonated histidylhistidine (HisHis): A model case for polyhistidyl-metal binding motifs

    NASA Astrophysics Data System (ADS)

    Peckelsen, Katrin; Martens, Jonathan; Berden, Giel; Oomens, Jos; Dunbar, Robert C.; Meijer, Anthony J. H. M.; Schäfer, Mathias

    2017-02-01

    In the complex formed between the calcium cation (Ca2+) and a deprotonated HisHis dipeptide, the complex adopts a charge solvation (CS) structure. Ca2+, a weak binding main group metal cation, interacts with the oxygens of the peptide carbonyl moiety and the deprotonated C-terminus. In contrast, the much stronger binding Ni2+ cation deprotonates the peptide nitrogen and induces an iminolate (Im) ligand structure in the [Ni(HisHis-H)]+ complex ion. The combination of infrared multiple-photon dissociation (IRMPD) spectroscopy and quantum chemistry evidence these two representative binding motifs. The iminolate coordination pattern identified and characterized in the [Ni(HisHis-H)]+ complex serves as a model case for nickel complexes of poly-histidyl-domains and is thereby also of interest to better understand the fundamentals of immobilized metal ion affinity chromatography as well as of Ni co-factor chemistry in enzymology.

  17. Nicotine induced CpG methylation of Pax6 binding motif in StAR promoter reduces the gene expression and cortisol production

    SciTech Connect

    Wang, Tingting; Chen, Man; Liu, Lian; Cheng, Huaiyan; Yan, You-E; Feng, Ying-Hong; Wang, Hui

    2011-12-15

    Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a single site CpG methylation at nt -377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming. -- Highlights: Black-Right-Pointing-Pointer Nicotine-induced StAR inhibition in two human adrenal cell models. Black-Right-Pointing-Pointer Nicotine-induced single CpG site methylation in StAR promoter. Black-Right-Pointing-Pointer Persistent StAR inhibition and single CpG methylation after nicotine termination

  18. Definition of fibronectin-mediated uptake of gelatinized latex by liver slices and macrophages.

    PubMed

    Molnar, J; Galles, M; Beezhold, D; Lai, M Z; Ku, C S; Van Alten, P J

    1987-10-01

    These studies show that both liver slices and macrophages carried out fibronectin concentration-dependent uptake of 125I-labeled gelatin-coated latex (test latex). Lack of phagocytosis of test latex by liver slices was shown directly by electron microscopy and indirectly by trypsin treatment, which caused the release of all test latex taken up in response to fibronectin. Inhibitors of phagocytosis did not alter this uptake. On the other hand, trypsin released only a portion of test latex from macrophages. Inhibitors of phagocytosis did not effect the released radioactive particles from macrophages but greatly reduced the trypsin-resistant radioactivity, taken as representing phagocytized particles. Opsonization of test latex with fibronectin did not require heparin but its association with liver slices occurred only in the presence of heparin. Macrophages, however, readily bound and internalized the opsonized test latex and heparin only potentiated these reactions. Gelatin competed with test latex for fibronectin for opsonization, but did not inhibit binding and phagocytosis of fibronectin-test latex complexes. Finally, soluble fibronectin-gelatin complexes did not compete for binding and phagocytosis of fibronectin-test latex complexes. Thus, fibronectin concentrated on the surface of latex is preferred for interaction with the fibronectin receptor of macrophages. Gelatin, however, was not essential for this reaction, because fibronectin directly coupled to latex was also readily taken up.

  19. Fibronectin in multiple sclerosis lesions.

    PubMed Central

    Sobel, R. A.; Mitchell, M. E.

    1989-01-01

    Cryostat sections of central nervous system (CNS) tissues of patients with multiple sclerosis (MS) and other CNS diseases were stained with antibodies to fibronectin, a macrophage fibronectin receptor component, fibrin/fibrinogen, and albumin using immunoperoxidase. In active, but not inactive, MS plaques vessel fibronectin was increased (to approximately 57% of Factor VIII+ vessels) over uninvolved MS and normal control white matter (P less than 0.001 for both). Fibronectin was primarily localized to vessel walls and amount of staining correlated with degree of inflammation. Active plaques and necrotic lesions also had extracellular fibronectin and fibrin/ogen. These molecules and the fibronectin receptor were found on macrophages. Albumin was more widely and diffusely distributed in lesions than fibronectin. Thus, in addition to extravasation from damaged vessels, fibronectin may be deposited on or synthesized by endothelial cells and macrophages in the CNS. Fibronectin could facilitate monocyte adhesion to endothelial cell luminal surfaces, promote migration of mononuclear cells, and enhance myelin phagocytosis in MS lesions. Images Figure 1 Figure 2 PMID:2528301

  20. The Role of Glutamic or Aspartic Acid in Position Four of the Epitope Binding Motif and Thyrotropin Receptor-Extracellular Domain Epitope Selection in Graves' Disease

    PubMed Central

    Inaba, Hidefumi; Martin, William; Ardito, Matt; De Groot, Anne Searls; De Groot, Leslie J.

    2010-01-01

    Context: Development of Graves' disease (GD) is related to HLA-DRB1*0301 (DR3),and more specifically to arginine at position 74 of the DRB1 molecule. The extracellular domain (ECD) of human TSH receptor (hTSH-R) contains the target antigen. Objective and Design: We analyzed the relation between hTSH-R-ECD peptides and DR molecules to determine whether aspartic acid (D) or glutamic acid (E) at position four in the binding motif influenced selection of functional epitopes. Results: Peptide epitopes from TSH-R-ECD with D or E in position four (D/E+) had higher affinity for binding to DR3 than peptides without D/E (D/E−) (IC50 29.3 vs. 61.4, P = 0.0024). HLA-DR7, negatively correlated with GD, and DRB1*0302 (HLA-DR18), not associated with GD, had different profiles of epitope binding. Toxic GD patients who are DR3+ had higher responses to D/E+ peptides than D/E− peptides (stimulation index 1.42 vs. 1.22, P = 0.028). All DR3+ GD patients (toxic + euthyroid) had higher responses, with borderline significance (Sl; 1.32 vs. 1.18, P = 0.051). Splenocytes of DR3 transgenic mice immunized to TSH-R-ECD responded to D/E+ peptides more than D/E− peptides (stimulation index 1.95 vs. 1.69, P = 0.036). Seven of nine hTSH-R-ECD peptide epitopes reported to be reactive with GD patients' peripheral blood mononuclear cells contain binding motifs with D/E at position four. Conclusions: TSH-R-ECD epitopes with D/E in position four of the binding motif bind more strongly to DRB1*0301 than epitopes that are D/E− and are more stimulatory to GD patients' peripheral blood mononuclear cells and to splenocytes from mice immunized to hTSH-R. These epitopes appear important in immunogenicity to TSH-R due to their favored binding to HLA-DR3, thus increasing presentation to T cells. PMID:20392871

  1. The Arabidopsis GAGA-Binding Factor BASIC PENTACYSTEINE6 Recruits the POLYCOMB-REPRESSIVE COMPLEX1 Component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA Motifs1

    PubMed Central

    Hecker, Andreas; Brand, Luise H.; Peter, Sébastien; Simoncello, Nathalie; Kilian, Joachim; Gaudin, Valérie

    2015-01-01

    Polycomb-repressive complexes (PRCs) play key roles in development by repressing a large number of genes involved in various functions. Much, however, remains to be discovered about PRC-silencing mechanisms as well as their targeting to specific genomic regions. Besides other mechanisms, GAGA-binding factors in animals can guide PRC members in a sequence-specific manner to Polycomb-responsive DNA elements. Here, we show that the Arabidopsis (Arabidopsis thaliana) GAGA-motif binding factor protein BASIC PENTACYSTEINE6 (BPC6) interacts with LIKE HETEROCHROMATIN PROTEIN1 (LHP1), a PRC1 component, and associates with VERNALIZATION2 (VRN2), a PRC2 component, in vivo. By using a modified DNA-protein interaction enzyme-linked immunosorbant assay, we could show that BPC6 was required and sufficient to recruit LHP1 to GAGA motif-containing DNA probes in vitro. We also found that LHP1 interacts with VRN2 and, therefore, can function as a possible scaffold between BPC6 and VRN2. The lhp1-4 bpc4 bpc6 triple mutant displayed a pleiotropic phenotype, extreme dwarfism and early flowering, which disclosed synergistic functions of LHP1 and group II plant BPC members. Transcriptome analyses supported this synergy and suggested a possible function in the concerted repression of homeotic genes, probably through histone H3 lysine-27 trimethylation. Hence, our findings suggest striking similarities between animal and plant GAGA-binding factors in the recruitment of PRC1 and PRC2 components to Polycomb-responsive DNA element-like GAGA motifs, which must have evolved through convergent evolution. PMID:26025051

  2. Cooperative Effects of Fibronectin Matrix Assembly and Initial Cell-Substrate Adhesion Strength in Cellular Self-Assembly

    PubMed Central

    Brennan, James R.; Hocking, Denise C.

    2015-01-01

    The cell-dependent polymerization of intercellular fibronectin fibrils can stimulate cells to self-assemble into multicellular structures. The local physical cues that support fibronectin-mediated cellular self-assembly are largely unknown. Here, fibronectin matrix analogs were used as synthetic adhesive substrates to model cell-matrix fibronectin fibrils having different integrin-binding specificity, affinity, and/or density. We utilized this model to quantitatively assess the relationship between adhesive forces derived from cell-substrate interactions and the ability of fibronectin fibril assembly to induce cellular self-assembly. Results indicate that the strength of initial, rather than mature, cell-substrate attachments correlates with the ability of substrates to support fibronectin-mediated cellular self-assembly. The cellular response to soluble fibronectin was bimodal and independent of the integrin-binding specificity of the substrate; increasing soluble fibronectin levels above a critical threshold increased aggregate cohesion on permissive substrates. Once aggregates formed, continuous fibronectin polymerization was necessary to maintain cohesion. During self-assembly, soluble fibronectin decreased cell-substrate adhesion strength and induced aggregate cohesion via a Rho-dependent mechanism, suggesting that the balance of contractile forces derived from fibronectin fibrils within cell-cell versus cell-substrate adhesions controls self-assembly and aggregate cohesion. Thus, initial cell-substrate attachment strength may provide a quantitative basis with which to build predictive models of fibronectin-mediated microtissue fabrication on a variety of substrates. PMID:26712598

  3. Redundant ERF-VII Transcription Factors Bind to an Evolutionarily Conserved cis-Motif to Regulate Hypoxia-Responsive Gene Expression in Arabidopsis

    PubMed Central

    Gasch, Philipp; Fundinger, Moritz; Müller, Jana T.; Lee, Travis; Mustroph, Angelika

    2016-01-01

    The response of Arabidopsis thaliana to low-oxygen stress (hypoxia), such as during shoot submergence or root waterlogging, includes increasing the levels of ∼50 hypoxia-responsive gene transcripts, many of which encode enzymes associated with anaerobic metabolism. Upregulation of over half of these mRNAs involves stabilization of five group VII ethylene response factor (ERF-VII) transcription factors, which are routinely degraded via the N-end rule pathway of proteolysis in an oxygen- and nitric oxide-dependent manner. Despite their importance, neither the quantitative contribution of individual ERF-VIIs nor the cis-regulatory elements they govern are well understood. Here, using single- and double-null mutants, the constitutively synthesized ERF-VIIs RELATED TO APETALA2.2 (RAP2.2) and RAP2.12 are shown to act redundantly as principle activators of hypoxia-responsive genes; constitutively expressed RAP2.3 contributes to this redundancy, whereas the hypoxia-induced HYPOXIA RESPONSIVE ERF1 (HRE1) and HRE2 play minor roles. An evolutionarily conserved 12-bp cis-regulatory motif that binds to and is sufficient for activation by RAP2.2 and RAP2.12 is identified through a comparative phylogenetic motif search, promoter dissection, yeast one-hybrid assays, and chromatin immunopurification. This motif, designated the hypoxia-responsive promoter element, is enriched in promoters of hypoxia-responsive genes in multiple species. PMID:26668304

  4. Central Role of the Copper-Binding Motif in the Complex Mechanism of Action of Ixosin: Enhancing Oxidative Damage and Promoting Synergy with Ixosin B.

    PubMed

    Libardo, M Daben J; Gorbatyuk, Vitaliy Y; Angeles-Boza, Alfredo M

    2016-01-08

    Ticks transmit multiple pathogens to different hosts without compromising their health. Their ability to evade microbial infections is largely a result of their effective innate immune response including various antimicrobial peptides. Therefore, a deep understanding of how ticks (and other arthropod vectors) control microbial loads could lead to the design of broad-spectrum antimicrobial agents. In this paper we study the role of the amino-terminal copper and nickel (ATCUN)-binding sequence in the peptide ixosin, isolated from the salivary glands of the hard tick Ixodes sinensis. Our results indicate that the ATCUN motif is not essential to the potency of ixosin, but is indispensable to its oxidative mechanism of action. Specifically, the ATCUN motif promotes dioxygen- and copper-dependent lipid (per)oxidation of bacterial membranes in a temporal fashion coinciding with the onset of bacterial death. Microscopy and studies on model membranes indicate that the oxidized phospholipids are utilized as potential targets of ixosin B (another tick salivary gland peptide) involving its delocalization to the bacterial membrane, thus resulting in a synergistic effect. Our proposed mechanism of action highlights the centrality of the ATCUN motif to ixosin's mechanism of action and demonstrates a novel way in which (tick) antimicrobial peptides (AMPs) utilize metal ions in its activity. This study suggests that ticks employ a variety of effectors to generate an amplified immune response, possibly justifying its vector competence.

  5. A polybasic motif in ErbB3-binding protein 1 (EBP1) has key functions in nucleolar localization and polyphosphoinositide interaction

    PubMed Central

    Karlsson, Thomas; Altankhuyag, Altanchimeg; Dobrovolska, Olena; Turcu, Diana C.; Lewis, Aurélia E.

    2016-01-01

    Polyphosphoinositides (PPIns) are present in the nucleus where they participate in crucial nuclear processes, such as chromatin remodelling, transcription and mRNA processing. In a previous interactomics study, aimed to gain further insight into nuclear PPIns functions, we identified ErbB3 binding protein 1 (EBP1) as a potential nuclear PPIn-binding protein in a lipid pull-down screen. EBP1 is a ubiquitous and conserved protein, located in both the cytoplasm and nucleolus, and associated with cell proliferation and survival. In the present study, we show that EBP1 binds directly to several PPIns via two distinct PPIn-binding sites consisting of clusters of lysine residues and positioned at the N- and C-termini of the protein. Using interaction mutants, we show that the C-terminal PPIn-binding motif contributes the most to the localization of EBP1 in the nucleolus. Importantly, a K372N point mutation, located within the C-terminal motif and found in endometrial tumours, is sufficient to alter the nucleolar targeting of EBP1. Our study reveals also the presence of the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110β and its product PtdIns(3,4,5)P3 together with EBP1 in the nucleolus. Using NMR, we further demonstrate an association between EBP1 and PtdIns(3,4,5)P3 via both electrostatic and hydrophobic interactions. Taken together, these results show that EBP1 interacts directly with PPIns and associate with PtdIns(3,4,5)P3 in the nucleolus. The presence of p110β and PtdIns(3,4,5)P3 in the nucleolus indicates their potential role in regulating nucleolar processes, at least via EBP1. PMID:27118868

  6. Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins.

    PubMed

    Koide, Takaki; Nishikawa, Yoshimi; Asada, Shinichi; Yamazaki, Chisato M; Takahara, Yoshifumi; Homma, Daisuke L; Otaka, Akira; Ohtani, Katsuki; Wakamiya, Nobutaka; Nagata, Kazuhiro; Kitagawa, Kouki

    2006-04-21

    The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa(-3)) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg(0)) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa(-3) and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa(-3) determined the binding affinity for HSP47. Maximal binding was observed when Yaa(-3) was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr(-3) and one Arg(0). The results revealed that HSP47 recognizes the Yaa(-3) and Arg(0) residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47.

  7. Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

    PubMed

    Cho, Ha Yeon; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Eungbin; Kang, Beom Sik

    2013-05-03

    The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.

  8. Identification of small-molecule inhibitors of calcineurin-NFATc signaling that mimic the PxIxIT motif of calcineurin binding partners.

    PubMed

    Matsoukas, Minos-Timotheos; Aranguren-Ibáñez, Álvaro; Lozano, Teresa; Nunes, Virginia; Lasarte, Juan José; Pardo, Leonardo; Pérez-Riba, Mercè

    2015-06-23

    Calcineurin (CN), a serine and threonine protein phosphatase that depends on Ca(2+) and calmodulin for its activity, is the target of the immunosuppressant drugs cyclosporin A (CsA) and tacrolimus (FK506). CN dephosphorylates and activates members of the NFATc (nuclear factor of activated T cells) family of transcription factors in T cells by binding to their conserved PxIxIT motif. Upon dephosphorylation, NFATc proteins translocate to the nucleus, where they stimulate the expression of genes encoding cytokines and chemokines that are required for T cell proliferation and the immune response. We performed a pharmacophore-based virtual screening of ~5.5 million commercially available, "drug-like" compounds to identify nonpeptidic compounds that inhibited the CN-dependent activation of NFATc signaling and that could serve as potential drug candidates for immunosuppressive therapy. Of 32 compounds that mimicked the PxIxIT motif, 7 competed with NFATc for binding to CN in vitro without interfering with the phosphatase activity of CN. Furthermore, in activated human CD4(+) T cells, four of the seven compounds inhibited the expression of NFATc-dependent genes, cytokine production, and cell proliferation, suggesting that these may have therapeutic potential as immunosuppressive agents.

  9. PDZ binding motif of HTLV-1 Tax promotes virus-mediated T-cell proliferation in vitro and persistence in vivo.

    PubMed

    Xie, Li; Yamamoto, Brenda; Haoudi, Abdelali; Semmes, O John; Green, Patrick L

    2006-03-01

    HTLV-1 cellular transformation and disease induction is dependent on expression of the viral Tax oncoprotein. PDZ is a modular protein interaction domain used in organizing signaling complexes in eukaryotic cells through recognition of a specific binding motif in partner proteins. Tax-1, but not Tax-2, contains a PDZ-binding domain motif (PBM) that promotes the interaction with several cellular PDZ proteins. Herein, we investigate the contribution of the Tax-1 PBM in HTLV-induced proliferation and immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. We generated several HTLV-1 and HTLV-2 Tax viral mutants, including HTLV-1deltaPBM, HTLV-2+C22(+PBM), and HTLV-2+ C18(deltaPBM). All Tax mutants maintained the ability to significantly activate the CREB/ATF or NFkappaB signaling pathways. Microtiter proliferation assays revealed that the Tax-1 PBM significantly increases both HTLV-1- and HTLV-2-induced primary T-cell proliferation. In addition, Tax-1 PBM was responsible for the micronuclei induction activity of Tax-1 relative to that of Tax-2. Viral infection and persistence were severely attenuated in rabbits inoculated with HTLV-1deltaPBM. Our results provide the first direct evidence suggesting that PBM-mediated associations between Tax-1 and cellular proteins play a key role in HTLV-induced cell proliferation and genetic instability in vitro and facilitate viral persistence in vivo.

  10. Bioassaying Putative RNA-Binding Motifs in a Protein Encoded by a Gene That Influences Courtship and Visually Mediated Behavior in Drosophila: In Vitro Mutagenesis of Nona

    PubMed Central

    Stanewsky, R.; Fry, T. A.; Reim, I.; Saumweber, H.; Hall, J. C.

    1996-01-01

    The no-on-transient-A (nonA) gene of Drosophila melanogaster influences vision, courtship song, and viability. The nonA-encoded polypeptide is inferred to bind single-stranded nucleic acids. Although sequence-analysis of NONA implies that it belongs to a special interspecific family of this protein type, it does contain two classical RNA recognition motifs (RRM). Their behavioral significance was assayed by generating transgenic strains that were singly or multiply mutated within the relatively N-terminal motif (RRM1) or within RRM2. Neither class of mutation affected NONA binding to polytene chromosomes. The former mutations led to extremely low viability, accompanied by diminished adult longevities that were much worse than for a nonA-null mutant, implying that faulty interpolypeptide interactions might accompany the effects of the amino-acid substitutions within RRM1. All in vitro-mutated types caused optomotor blindness and an absence of transient spikes in the electroretinogram. Courtship analysis discriminated between the effects of the mutations: the RRM2-mutated type generated song pulses and trains that tended to be mildly mutant. These phenotypic abnormalities reinforce the notion that nonA's ubiquitous expression has its most important consequences in the optic lobes, the thoracic ganglia, or both, depending in part on the nonA allele. PMID:8722780

  11. Allosteric regulation of helicase core activities of the DEAD-box helicase YxiN by RNA binding to its RNA recognition motif.

    PubMed

    Samatanga, Brighton; Andreou, Alexandra Z; Klostermeier, Dagmar

    2017-01-23

    DEAD-box proteins share a structurally similar core of two RecA-like domains (RecA_N and RecA_C) that contain the conserved motifs for ATP-dependent RNA unwinding. In many DEAD-box proteins the helicase core is flanked by ancillary domains. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA recognition motif (RRM), we investigated the effect of RNA binding to the RRM on its position relative to the core, and on core activities. RRM/RNA complex formation substantially shifts the RRM from a position close to the RecA_C to the proximity of RecA_N, independent of RNA contacts with the core. RNA binding to the RRM is communicated to the core, and stimulates ATP hydrolysis and RNA unwinding. The conformational space of the core depends on the identity of the RRM-bound RNA. Allosteric regulation of core activities by RNA-induced movement of ancillary domains may constitute a general regulatory mechanism of DEAD-box protein activity.

  12. ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting.

    PubMed

    Dores, Michael R; Chen, Buxin; Lin, Huilan; Soh, Unice J K; Paing, May M; Montagne, William A; Meerloo, Timo; Trejo, JoAnn

    2012-04-30

    The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs.

  13. DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy

    PubMed Central

    Tubbs, Julie L.; Pegg, Anthony E.; Tainer, John A.

    2007-01-01

    O6-alkylguanine-DNA alkyltransferase (AGT) is a crucial target both for the prevention of cancer and for chemotherapy, since it repairs mutagenic lesions in DNA, and it limits the effectiveness of alkylating chemotherapies. AGT catalyzes the unique, single-step, direct damage reversal repair of O6-alkylguanines by selectively transferring the O6-alkyl adduct to an internal cysteine residue. Recent crystal structures of human AGT alone and in complex with substrate DNA reveal a two-domain a/β fold and a bound zinc ion. AGT uses its helix-turn-helix motif to bind substrate DNA via the minor groove. The alkylated guanine is then flipped out from the base stack into the AGT active site for repair by covalent transfer of the alkyl adduct to Cys145. An asparagine hinge (Asn137) couples the helix-turn-helix DNA binding and active site motifs. An arginine finger (Arg128) stabilizes the extrahelical DNA conformation. With this newly improved structural understanding of AGT and its interactions with biologically relevant substrates, we can now begin to unravel the role it plays in preserving genetic integrity and discover how it promotes resistance to anticancer therapies. PMID:17485252

  14. The PPFLMLLKGSTR motif in globular domain 3 of the human laminin-5 {alpha}3 chain is crucial for integrin {alpha}3{beta}1 binding and cell adhesion

    SciTech Connect

    Kim, Jin-Man; Park, Won Ho; Min, Byung-Moo . E-mail: bmmin@snu.ac.kr

    2005-03-10

    Laminin-5 regulates various cellular functions, including cell adhesion, spreading, and motility. Here, we expressed the five human laminin {alpha}3 chain globular (LG) domains as monomeric, soluble fusion proteins, and examined their biological functions and signaling. Recombinant LG3 (rLG3) protein, unlike rLG1, rLG2, rLG4, and rLG5, played roles in cell adhesion, spreading, and integrin {alpha}3{beta}1 binding. More significantly, we identified a novel motif (PPFLMLLKGSTR) in the LG3 domain that is crucial for these responses. Studies with the synthetic peptides delineated the PPFLMLLKGSTR peptide within LG3 domain as a major site for both integrin {alpha}3{beta}1 binding and cell adhesion. Substitution mutation experiments suggest that the Arg residue is important for these activities. rLG3 protein- and PPFLMLLKGSTR peptide-induced keratinocyte adhesion triggered cell signaling through FAK phosphorylation at tyrosine-397 and -577. To our knowledge, this is the first report demonstrating that the PPFLMLLKGSTR peptide within the LG3 domain is a novel motif that is capable of supporting integrin {alpha}3{beta}1-dependent cell adhesion and spreading.

  15. Crystallization and preliminary crystallographic analysis of calcium-binding protein-2 from Entamoeba histolytica and its complexes with strontium and the IQ1 motif of myosin V

    SciTech Connect

    Gourinath, S. Padhan, Narendra; Alam, Neelima; Bhattacharya, Alok

    2005-04-01

    Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Two domains with four canonical EF-hand-containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though they have very high sequence similarity, these bind to different target proteins in a Ca{sup 2+}-dependent manner, leading to different functional pathways. Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 111.74, b = 68.83, c = 113.25 Å, β = 116.7°. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 69.18, b = 112.03, c = 93.42 Å, β = 92.8°. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. This complex was crystallized with MPD and ethanol as precipitating agents. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 60.5, b = 69.86, c = 86.5 Å, β = 97.9°.

  16. Trypanosoma cruzi Binds to Cytokeratin through Conserved Peptide Motifs Found in the Laminin-G-Like Domain of the gp85/Trans-sialidase Proteins

    PubMed Central

    Teixeira, Andre Azevedo Reis; de Vasconcelos, Veronica de Cássia Sardinha; Colli, Walter; Alves, Maria Júlia Manso; Giordano, Ricardo José

    2015-01-01

    Background Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a disease that affects millions of people most of them living in South and Central Americas. There are few treatment options for individuals with Chagas' disease making it important to understand the molecular details of parasite infection, so novel therapeutic alternatives may be developed for these patients. Here, we investigate the interaction between host cell intermediate filament proteins and the T. cruzi gp85 glycoprotein superfamily with hundreds of members that have long been implicated in parasite cell invasion. Methodology/Principal Findings An in silico analysis was utilized to identify peptide motifs shared by the gp85 T. cruzi proteins and, using phage display, these selected peptide motifs were screened for their ability to bind to cells. One peptide, named TS9, showed significant cell binding capacity and was selected for further studies. Affinity chromatography, phage display and invasion assays revealed that peptide TS9 binds to cytokeratins and vimentin, and prevents T. cruzi cell infection. Interestingly, peptide TS9 and a previously identified binding site for intermediate filament proteins are disposed in an antiparallel β-sheet fold, present in a conserved laminin-G-like domain shared by all members of the family. Moreover, peptide TS9 overlaps with an immunodominant T cell epitope. Conclusions/Significance Taken together, the present study reinforces previous results from our group implicating the gp85 superfamily of glycoproteins and the intermediate filament proteins cytokeratin and vimentin in the parasite infection process. It also suggests an important role in parasite biology for the conserved laminin-G-like domain, present in all members of this large family of cell surface proteins. PMID:26398185

  17. Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles.

    PubMed Central

    Chen, Y; Ludescher, R D; Montville, T J

    1997-01-01

    The purpose of this study was to characterize in detail the binding of pediocin PA-1 and its fragments to target membranes by using tryptophan fluorescence as a probe. Based on a three-dimensional model (Y. Chen, R. Shapira, M. Eisenstein, and T. J. Montville, Appl. Environ. Microbiol. 63:524-531, 1997), four synthetic N-terminal pediocin fragments were selected to study the mechanism of the initial step by which the bacteriocin associates with membranes. Binding of pediocin PA-1 to vesicles of phosphatidylglycerol, the major component of Listeria membranes, caused an increase in the intrinsic tryptophan fluorescence intensity with a blue shift of the emission maximum. The Stern-Volmer constants for acrylamide quenching of the fluorescence of pediocin PA-1 in buffer and in the lipid vesicles were 8.83 +/- 0.42 and 3.53 +/- 0.67 M-1, respectively, suggesting that the tryptophan residues inserted into the hydrophobic core of the lipid bilayer. The synthetic pediocin fragments bound strongly to the lipid vesicles when a patch of positively charged amino acid residues (K-11 and H-12) was present but bound weakly when this patch was mutated out. Quantitative comparison of changes in tryptophan fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the relative affinity to the lipid vesicles paralleled the net positive charge in the peptide. The relative affinity for the fragment containing the YGNGV consensus motif was 10-fold lower than that for the fragment containing the positive patch. Furthermore, changing the pH from 6.0 to 8.0 decreased binding of the fragments containing the positive patch, probably due to deprotonation of His residues. These results demonstrate that electrostatic interactions, but not the YGNGV motif, govern pediocin binding to the target membrane. PMID:9406395

  18. Tendon synovial cells secrete fibronectin in vivo and in vitro

    SciTech Connect

    Banes, A.J.; Link, G.W.; Bevin, A.G.; Peterson, H.D.; Gillespie, Y.; Bynum, D.; Watts, S.; Dahners, L.

    1988-01-01

    The chemistry and cell biology of the tendon have been largely overlooked due to the emphasis on collagen, the principle structural component of the tendon. The tendon must not only transmit the force of muscle contraction to bone to effect movement, but it must also glide simultaneously over extratendonous tissues. Fibronectin is classified as a cell attachment molecule that induces cell spreading and adhesion to substratum. The external surface of intact avian flexor tendon stained positively with antibody to cellular fibronectin. However, if the surface synovial cells were first removed with collagenase, no positive reaction with antifibronectin antibody was detected. Analysis of immunologically stained frozen sections of tendon also revealed fibronectin at the tendon synovium, but little was associated with cells internal in tendon. The staining pattern with isolated, cultured synovial cells and fibroblasts from the tendon interior substantiated the histological observations. Analysis of polyacrylamide gel profiles of /sup 35/S-methionine-labeled proteins synthesized by synovial cells and internal fibroblasts indicated that fibronectin was synthesized principally by synovial cells. Fibronectin at the tendon surface may play a role in cell attachment to prevent cell removal by the friction of gliding. Alternatively, fibronectin, with its binding sites for hyaluronic acid and collagen, may act as a complex for boundary lubrication.

  19. A rapid method for fibronectin purification on nitrocellulose membranes suitable for tissue culture.

    PubMed

    Chifflet, Silvia; Bolatto, Carmen; Tolosa, Susana

    2004-05-31

    We have developed a simple method for plasma fibronectin purification based on the well-known gelatin binding property of fibronectin. In this procedure we immobilize the melted gelatin to nitrocellulose membranes; these are then used to affinity-purify the fibronectin from the plasma sample. The fibronectin is eluted from the membrane by treatment with 8 M urea. The procedure described here gives a yield of up to 60% (from presumed fibronectin concentration) and the fibronectin obtained is homogeneous in SDS-PAGE and biologically active, as assessed by a cell migration assay. The method is rapid, simple, inexpensive, does not require the use of chromatographic equipment and is suitable for tissue culture applications.

  20. Fibronectin changes in eosinophilic meningitis with blood-CSF barrier disruption.

    PubMed

    Shyu, Ling-Yuh; Hu, Ming-E; Chou, Chun-Hui; Chen, Ke-Min; Chiu, Ping-Sung; Lai, Shih-Chan

    2015-01-01

    Fibronectin, which is present at relatively low levels in healthy central nervous systems (CNS), shows increased levels in meningitis. In this study, fibronectin processing was correlated with the increased permeability of the blood-cerebrospinal fluid (CSF) barrier as well as with the formation of eosinophil infiltrates in angiostrongyliasis meningitis. The immunohistochemistry results show matrix metalloproteinase-9 (MMP-9) is localized in the choroid plexus epithelium. Coimmunoprecipitation demonstrated fibronectin strongly binds MMP-9. Furthermore, treatment with the MMP-9 inhibitor GM6001 significantly inhibited fibronectin processing, reduced the blood-CSF barrier permeability, and decreased the eosinophil counts. The decreased fibronectin processing in CSF implies decreased cellular invasion of the subarachnoid space across the blood-CSF barrier. Therefore, increased fibronectin processing may be associated with barrier disruption and participate in the extravasation and migration of eosinophils into the CNS during experimental parasitic infection.

  1. Delayed secondary glucocorticoid response elements. Unusual nucleotide motifs specify glucocorticoid receptor binding to transcribed regions of alpha 2u-globulin DNA.

    PubMed

    Chan, G C; Hess, P; Meenakshi, T; Carlstedt-Duke, J; Gustafsson, J A; Payvar, F

    1991-11-25

    Glucocorticoids stimulate the transcription of rat alpha 2u-globulin (RUG) genes. Because this induction occurs after a time lag of several hours and is blocked by inhibitors of protein synthesis, it exemplifies a delayed secondary response to steroid hormones. In this report, we show that a region of RUG-transcribed DNA (approximately +1800 to +2174) contains multiple footprint sites for glucocorticoid receptor that are, apparently, organized into at least three independent binding clusters. The DNA sequences bound by the receptor and the location of binding sites were determined. A family of sequences related to half-sites of the consensus primary glucocorticoid response element (GRE) is discernible at each cluster of sites. Compared to the consensus GRE, which contains two pseudo-palindromic hexanucleotides arranged in a tail-to-tail fashion and separated by three bases, the arrangements of hexanucleotides within this segment of RUG DNA are unusual and heterogeneous. Methylation interference of a binding cluster containing three receptor footprints demonstrates that certain guanines of the GRE-like hexanucleotides are essential for efficient receptor binding. A synthetic 29-base pair (bp) RUG element, containing one receptor footprint from this cluster, selectively binds the receptor. Within this 29-bp element, six nucleotides separate two directly repeated copies of GRE-like hexanucleotides. RUG DNA fragments containing all or part of the three binding clusters, including the 29-bp element, confer a delayed secondary hormone responsiveness upon a linked heterologous promoter and reporter gene in stably transfected cell lines. We speculate that the unusual DNA sequence motifs of the receptor-binding sites are crucial for the generation of certain delayed secondary responses.

  2. A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins

    PubMed Central

    1995-01-01

    Many integrins recognize short RGD-containing amino acid sequences and such peptide sequences can be identified from phage libraries by panning with an integrin. Here, in a reverse strategy, we have used such libraries to isolate minimal receptor sequences that bind to fibronectin and RGD-containing fibronectin fragments in affinity panning. A predominant cyclic motif, *CWDDG/LWLC*, was obtained (the asterisks denote a potential disulfide bond). Studies using the purified phage and the corresponding synthetic cyclic peptides showed that *CWDDGWLC*-expressing phage binds specifically to fibronectin and to fibronectin fragments containing the RGD sequence. The binding did not require divalent cations and was inhibited by both RGD and *CWDDGWLC*-containing synthetic peptides. Conversely, RGD-expressing phage attached specifically to immobilized *CWDDGWLC*-peptide and the binding could be blocked by the respective synthetic peptides in solution. Moreover, fibronectin bound to a *CWDDGWLC*-peptide affinity column, and could be eluted with an RGD-containing peptide. The *CWDDGWLC*-peptide inhibited RGD-dependent cell attachment to fibronectin and vitronectin, but not to collagen. A region of the beta subunit of RGD-binding integrins that has been previously demonstrated to be involved in ligand binding includes a polypeptide stretch, KDDLW (in beta 3) similar to WDDG/LWL. Synthetic peptides corresponding to this region in beta 3 were found to bind RGD-displaying phage and conversion of its two aspartic residues into alanines greatly reduced the RGD binding. Polyclonal antibodies raised against the *CWDDGWLC*- peptide recognized beta 1 and beta 3 in immunoblots. These data indicate that the *CWDDGWLC*-peptide is a functional mimic of ligand binding sites of RGD-directed integrins, and that the structurally similar site in the integrin beta subunit is a binding site for RGD. PMID:7657703

  3. Mycoplasma hyopneumoniae Surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs.

    PubMed

    Deutscher, Ania T; Tacchi, Jessica L; Minion, F Chris; Padula, Matthew P; Crossett, Ben; Bogema, Daniel R; Jenkins, Cheryl; Kuit, Tracey A; Walker, Mark J; Djordjevic, Steven P

    2012-03-02

    P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site (761)L-N-V↓A-V-S(766) in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.

  4. Human macrophage differentiation involves an interaction between integrins and fibronectin

    SciTech Connect

    Laouar, A.; Chubb, C.B.H.; Collart, F.; Huberman, E.

    1996-11-15

    The authors have examined the role of the {beta}{sub 1} integrin family of adhesion receptors (VLA) and the extracellular matrix protein fibronectin (FN) in macrophage differentiation of (1) human HL-60 myeloid leukemia cells induced by phorbol 12-myristate 13-acetate (PMA) and (2) human peripheral blood monocytes induced by either PMA or macrophage-colony stimulating factor (M=CSF). Increased VLA and FN gene expression was observed as early as 4 h after PMA treatment of HL-60 cells and PMA- or M-CSF-treatment of monocytes, and it preceded the manifestation of macrophage markers. Treated HL-60 cells and monocytes also released and deposited FN on the surface of the tissue culture dishes. An HL-60 cell variant, HL-525, which is deficient in protein kinase C {beta} and resistant to PMA-induced differentiation, exhibited elevated levels of the VLA antigen but failed to express the FN gene. Incubation of HL-525 cells on dishes precoated with exogenous FN resulted in a macrophage differentiation. The macrophage phenotype induced in HL-60 cells, HL-525 cells, or monocytes was attenuated to various degrees by anti-VLA or anti-FN MAbs or by exogenous RGDS, a VLA-binding motif on FN. The authors suggest that macrophage differentiation is initiated by the activation of protein kinase C, which leads to the expression of the integrin, FN and related genes. The integrins mediate cell attachment and spreading on appropriate substrates by binding to deposited extracellular proteins such as FN. This attachment and spreading, in turn, leads to the expression of genes that code for the macrophage functions.

  5. Novel antiplatelet role for a protein disulfide isomerase-targeted peptide: Evidence of covalent binding to C-terminal CGHC redox motif.

    PubMed

    Sousa, H R; Gaspar, R S; Sena, E M L; da Silva, S A; de L Fontelles, J L; Araújo, T L S; Mastrogiovanni, M; Fries, D M; Azevedo-Santos, A P S; Laurindo, F R M; Trostchansky, A; Paes, A M

    2017-01-21

    Protein disulfide isomerase (PDI) plays a major role in platelet aggregation and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized this peptide would also inhibit platelet aggregation by association to surface PDI. Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. We showed that CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. In thrombin-activated platelets, surface expression of activated αIIbβ3 integrin, P-selectin and granulophysin were analyzed by flow cytometry. CxxC decreased αIIbβ3 activation, but had no effect on the other markers. Upon membrane free thiol labeling in resting platelets, CxxC decreased cell-surface PDI pull down without interfering in total thiol protein content. Finally, the capacity of CxxC to covalently bind to PDI active site was analyzed by ESI LC-MS/MS. We detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 , an important site for PDI prothrombotic activity. Interestingly, CxxC did not react with oxidized PDI. In summary, our data support an antiplatelet activity for CxxC through binding to Cys400 in PDI a' domain, which can be further exploited as a model for site-driven antithrombotic agents development. This article is protected by copyright. All rights reserved.

  6. Generation of mice deficient in RNA-binding motif protein 3 (RBM3) and characterization of its role in innate immune responses and cell growth

    SciTech Connect

    Matsuda, Atsushi; Ogawa, Masahiro; Yanai, Hideyuki; Naka, Daiji; Goto, Ayana; Ao, Tomoka; Tanno, Yuji; Takeda, Kiyoshi; Watanabe, Yoshinori; Honda, Kenya; Taniguchi, Tadatsugu

    2011-07-22

    Highlights: {yields} We identified RNA-binding motif protein 3 (RBM3) as CpG-B DNA-binding protein. {yields} RBM3 translocates from the nucleus to the cytoplasm and co-localized with CpG-B DNA. {yields} We newly generated Rbm3-deficient (Rbm3{sup -/-}) mice. {yields} DNA-mediated cytokine gene induction was normally occured in Rbm3{sup -/-} cells. {yields}Rbm3{sup -/-} MEFs showed poorer proliferation rate and increased number of G2-phase cells. -- Abstract: The activation of innate immune responses is critical to host defense against microbial infections, wherein nucleic acid-sensing pattern recognition receptors recognize DNA or RNA from viruses or bacteria and activate downstream signaling pathways. In a search for new DNA-sensing molecules that regulate innate immune responses, we identified RNA-binding motif protein 3 (RBM3), whose role has been implicated in the regulation of cell growth. In this study, we generated Rbm3-deficient (Rbm3{sup -/-}) mice to study the role of RBM3 in immune responses and cell growth. Despite evidence for its interaction with immunogenic DNA in a cell, no overt phenotypic abnormalities were found in cells from Rbm3{sup -/-} mice for the DNA-mediated induction of cytokine genes. Interestingly, however, Rbm3{sup -/-} mouse embryonic fibroblasts (MEFs) showed poorer proliferation rates as compared to control MEFs. Further cell cycle analysis revealed that Rbm3{sup -/-} MEFs have markedly increased number of G2-phase cells, suggesting a hitherto unknown role of RBM3 in the G2-phase control. Thus, these mutant mice and cells may provide new tools with which to study the mechanisms underlying the regulation of cell cycle and oncogenesis.

  7. FACT Disrupts Nucleosome Structure by Binding H2A-H2B with Conserved Peptide Motifs.

    PubMed

    Kemble, David J; McCullough, Laura L; Whitby, Frank G; Formosa, Tim; Hill, Christopher P

    2015-10-15

    FACT, a heterodimer of Spt16 and Pob3, is an essential histone chaperone. We show that the H2A-H2B binding activity that is central to FACT function resides in short acidic regions near the C termini of each subunit. Mutations throughout these regions affect binding and cause correlated phenotypes that range from mild to lethal, with the largest individual contributions unexpectedly coming from an aromatic residue and a nearby carboxylate residue within each domain. Spt16 and Pob3 bind overlapping sites on H2A-H2B, and Spt16-Pob3 heterodimers simultaneously bind two H2A-H2B dimers, the same stoichiometry as the components of a nucleosome. An Spt16:H2A-H2B crystal structure explains the biochemical and genetic data, provides a model for Pob3 binding, and implies a mechanism for FACT reorganization that we confirm biochemically. Moreover, unexpected similarity to binding of ANP32E and Swr1 with H2A.Z-H2B reveals that diverse H2A-H2B chaperones use common mechanisms of histone binding and regulating nucleosome functions.

  8. Myosin IIIB uses an actin-binding motif in its espin-1 cargo to reach the tips of actin protrusions.

    PubMed

    Merritt, Raymond C; Manor, Uri; Salles, Felipe T; Grati, M'hamed; Dose, Andrea C; Unrath, William C; Quintero, Omar A; Yengo, Christopher M; Kachar, Bechara

    2012-02-21

    Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.

  9. FACT disrupts nucleosome structure by binding H2A-H2B with conserved peptide motifs

    PubMed Central

    Kemble, David J.; McCullough, Laura L.; Whitby, Frank G.; Formosa, Tim; Hill, Christopher P.

    2015-01-01

    SUMMARY FACT, a heterodimer of Spt16 and Pob3, is an essential histone chaperone. We show that the H2A-H2B binding activity that is central to FACT function resides in short acidic regions near the C-termini of each subunit. Mutations throughout these regions impact binding and cause correlated phenotypes that range from mild to lethal, with the largest individual contributions unexpectedly coming from an aromatic residue and a nearby carboxylate residue within each domain. Spt16 and Pob3 bind overlapping sites on H2A-H2B, and Spt16-Pob3 heterodimers simultaneously bind two H2A-H2B dimers, the same stoichiometry as the components of a nucleosome. An Spt16:H2A-H2B crystal structure explains the biochemical and genetic data, provides a model for Pob3 binding, and implies a mechanism for FACT reorganization that we confirm biochemically. Moreover, unexpected similarity to binding of ANP32E and Swr1 with H2A.Z-H2B reveals that diverse H2A-H2B chaperones use common mechanisms of histone binding and regulating nucleosome functions. PMID:26455391

  10. Switch recombination breakpoints are strictly correlated with DNA recognition motifs for immunoglobulin S gamma 3 DNA-binding proteins

    PubMed Central

    1992-01-01

    The deletion looping out model of switch (S) recombination predicts that the intervening DNA between switch regions will be excised as a circle. Circular excision products of immunoglobulin switch recombination have been recently isolated from lipopolysaccharide (LPS)- stimulated spleen cells. The recombination breakpoints in these large circles were found to fall within switch regions. Since switch recombination is clearly focused on switch regions, we hypothesized that some DNA-binding protein factor might be involved in specifically recognizing and facilitating the alignment of switch regions before recombination. Two DNA-binding proteins that specifically interact with two discrete regions of the S gamma 3 tandem repeat have been identified in crude and partially purified nuclear extracts derived from LPS- and dextran sulfate (DxS)-activated splenic B cells. The first factor has been found indistinguishable from NF-kappa B by mobility shift assays, methylation interference, competition binding studies, and supershift analysis using an antiserum specific for the p50 component. The second appears to be composed of two closely traveling mobilities that do not separate upon partial purification. This second complex is unique and specific for S gamma 3 by methylation interference assays and competition-binding analysis. The sites at which recombination occurs in the S gamma 3 switch region have been analyzed and found to strictly correlate with the binding sites of the S gamma 3 switch binding proteins. PMID:1500850

  11. Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif.

    PubMed Central

    Sharrocks, A D; Gille, H; Shaw, P E

    1993-01-01

    The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure. Images PMID:8417320

  12. A fibronectin receptor on Candida albicans mediates adherence of the fungus to extracellular matrix

    SciTech Connect

    Klotz, S.A.; Smith, R.L. )

    1991-03-01

    Binding of fibronectin, an extracellular matrix (ECM) protein, to Candida albicans was measured, and adherence of the fungus to immobilized ECM proteins, fibronectin, laminin, types I and IV collagen, and subendothelial ECM was studied. 125I-labeled fibronectin was inhibited from binding to the fungus by unlabeled human plasma fibronectin and by Arg-Gly-Asp (RGD), Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), and Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP), but binding was not inhibited by Gly-Arg-Gly-Asp-Ser-Pro. Soluble fibronectin, RGD, GRGESP, and GRGDTP also inhibited fungal adherence to the individual immobilized ECM proteins in a complex pattern, but only soluble fibronectin (10(-7) M) inhibited fungal adherence to subendothelial ECM. Thus, C. albicans possesses at least one type of cell surface receptor for binding soluble fibronectin that can be inhibited with peptides. This receptor apparently is used to bind the fungus to immobilized ECM proteins and to subendothelial ECM and may play a role in the initiation of disseminated disease by bloodborne fungi by providing for adherence of the microorganisms to ECM proteins.

  13. Specific Ca2+-binding motif in the LH1 complex from photosynthetic bacterium Thermochromatium tepidum as revealed by optical spectroscopy and structural modeling.

    PubMed

    Ma, Fei; Kimura, Yukihiro; Yu, Long-Jiang; Wang, Peng; Ai, Xi-Cheng; Wang, Zheng-Yu; Zhang, Jian-Ping

    2009-03-01

    Native and Ca(2+)-depleted light-harvesting-reaction center core complexes (LH1-RC) from the photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibit maximal LH1-Q(y) absorption at 915 and 889 nm, respectively. To understand the structural origins of the spectral variation, we performed spectroscopic and structure modeling investigations. For the 889 nm form of LH1-RC, bacteriochlorophyll a (BChl a) in the native form was found by means of near-infrared Fourier-transform Raman spectroscopy, a higher degree of macrocycle distortion and a stronger hydrogen bond with the beta-Trp(-8) residue. SWISS-MODEL structure modeling suggests the presence of a specific coordination motif of Ca(2+) at the C-terminus of the alpha-subunit of LH1, while MODELLER reveals the tilt of alpha- and beta-polypeptides with reference to the structural template, as well as a change in the concentric orientation of BChl a molecules, both of which may be connected to the long-wavelength LH1-Q(y) absorption of the 915 nm form. The carotenoid spirilloxanthin shows a twisted all-trans configuration in both forms of LH1 as evidenced by the resonance Raman spectroscopic results. With regard to the thermal stability, the 915 nm form was shown by the use of temperature-dependent fluorescence spectroscopy to be approximately 20 K more stable than the 889 nm form, which may be ascribed to the specific Ca(2+)-binding motif of LH1.

  14. TFII-I regulates target genes in the PI-3K and TGF-β signaling pathways through a novel DNA binding motif.

    PubMed

    Segura-Puimedon, Maria; Borralleras, Cristina; Pérez-Jurado, Luis A; Campuzano, Victoria

    2013-09-25

    General transcription factor (TFII-I) is a multi-functional protein involved in the transcriptional regulation of critical developmental genes, encoded by the GTF2I gene located on chromosome 7q11.23. Haploinsufficiency at GTF2I has been shown to play a major role in the neurodevelopmental features of Williams-Beuren syndrome (WBS). Identification of genes regulated by TFII-I is thus critical to detect molecular determinants of WBS as well as to identify potential new targets for specific pharmacological interventions, which are currently absent. We performed a microarray screening for transcriptional targets of TFII-I in cortex and embryonic cells from Gtf2i mutant and wild-type mice. Candidate genes with altered expression were verified using real-time PCR. A novel motif shared by deregulated genes was found and chromatin immunoprecipitation assays in embryonic fibroblasts were used to document in vitro TFII-I binding to this motif in the promoter regions of deregulated genes. Interestingly, the PI3K and TGFβ signaling pathways were over-represented among TFII-I-modulated genes. In this study we have found a highly conserved DNA element, common to a set of genes regulated by TFII-I, and identified and validated novel in vivo neuronal targets of this protein affecting the PI3K and TGFβ signaling pathways. Overall, our data further contribute to unravel the complexity and variability of the different genetic programs orchestrated by TFII-I.

  15. Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

    NASA Astrophysics Data System (ADS)

    Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

    2013-03-01

    Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses

  16. Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence.

    PubMed

    Drayer, A L; Meima, M E; Derks, M W; Tuik, R; van Haastert, P J

    1995-10-15

    Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.

  17. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    PubMed

    Garbelli, Anna; Beermann, Sandra; Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-05-12

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  18. Purification, crystallization and preliminary crystallographic studies of C-terminal RNA recognition motif (RRM-3) of human ELAV-type RNA-binding protein 3 (ETR-3)

    PubMed Central

    Kashyap, Maruthi; Sharma, Ashwani; Bhavesh, Neel Sarovar

    2013-01-01

    Human embryonically lethal abnormal vision (ELAV)-type RNA-binding protein 3 (ETR-3) has been implicated in many aspects of RNA-processing events including alternative splicing, stability, editing and translation. RNA recognition motif 3 (RRM-3) is an independent C-terminal RNA-binding domain of ETR-3 that preferentially binds to UG-rich repeats of the nuclear or cytoplasmic pre-mRNA, and along with the other domains mediates the inclusion of cardiac troponin T (c-TNT) exon 5 in embryonic muscle, which is otherwise excluded in the adult. In the present study, RRM-3 was cloned, overexpressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 3 Å resolution at the home source and belonged to space group P213, with unit-cell parameters a = b = c = 118.5 Å, α = β = γ = 90°. There were two molecules of RRM-3 in the asymmetric unit and the calculated Matthews coefficient (V M) was 6.35 Å3 Da−1, with a solvent content of 80.62%. Initial phases were determined by molecular replacement. PMID:24100559

  19. A Small Chimeric Fibronectin Fragment Accelerates Dermal Wound Repair in Diabetic Mice

    PubMed Central

    Hocking, Denise C.; Brennan, James R.; Raeman, Carol H.

    2016-01-01

    Objective: During wound repair, soluble fibronectin is converted into biologically active, insoluble fibrils via a cell-mediated process. This fibrillar, extracellular matrix (ECM) form of fibronectin stimulates cell processes critical to tissue repair. Nonhealing wounds show reduced levels of ECM fibronectin fibrils. The objective of this study was to produce a small, recombinant wound supplement with the biological activity of insoluble fibronectin fibrils. Approach: A chimeric fibronectin fragment was produced by inserting the integrin-binding Arg-Gly-Asp (RGD) loop from the tenth type III repeat of fibronectin (FNIII10) into the analogous site within the heparin-binding, bioactive fragment of the first type III repeat (FNIII1H). FNIII1HRGD was tested for its ability to support cell functions necessary for wound healing, and then evaluated for its capacity to accelerate healing of full-thickness dermal wounds in diabetic mice. Results: In vitro, FNIII1HRGD supported cell adhesion, proliferation, and ECM fibronectin deposition. Application of FNIII1HRGD to dermal wounds of diabetic mice significantly enhanced wound closure compared with controls (73.9% ±4.1% vs. 58.1% ±4.7% closure on day 9, respectively), and significantly increased granulation tissue thickness (2.88 ± 0.75-fold increase over controls on day 14). Innovation: Recombinant proteins designed to functionally mimic the ECM form of fibronectin provide a novel therapeutic approach to circumvent diminished fibronectin fibril formation by delivering ECM fibronectin signals in a soluble form to chronic wounds. Conclusion: A small, chimeric fibronectin protein was developed. FNIII1HRGD demonstrated enhanced bioactivity in vitro and stimulated wound repair in a murine model of chronic wounds. PMID:27867754

  20. The structure of Plasmodium vivax phosphatidylethanolamine-binding protein suggests a functional motif containing a left-handed helix

    SciTech Connect

    Arakaki, Tracy; Neely, Helen; Boni, Erica; Mueller, Natasha; Buckner, Frederick S.; Van Voorhis, Wesley C.; Lauricella, Angela; DeTitta, George; Luft, Joseph; Hol, Wim G. J.; Merritt, Ethan A.

    2007-03-01

    The crystal structure of a phosphatidylethanolamine-binding protein from P. vivax, a homolog of Raf-kinase inhibitor protein (RKIP), has been solved to a resolution of 1.3 Å. The inferred interaction surface near the anion-binding site is found to include a distinctive left-handed α-helix. The structure of a putative Raf kinase inhibitor protein (RKIP) homolog from the eukaryotic parasite Plasmodium vivax has been studied to a resolution of 1.3 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protozoan protein is topologically similar to previously studied members of the phosphatidylethanolamine-binding protein (PEBP) sequence family, but exhibits a distinctive left-handed α-helical region at one side of the canonical phospholipid-binding site. Re-examination of previously determined PEBP structures suggests that the P. vivax protein and yeast carboxypeptidase Y inhibitor may represent a structurally distinct subfamily of the diverse PEBP-sequence family.

  1. Two-dimensional combinatorial screening of a bacterial rRNA A-site-like motif library: defining privileged asymmetric internal loops that bind aminoglycosides.

    PubMed

    Tran, Tuan; Disney, Matthew D

    2010-03-09

    RNAs have diverse structures that are important for biological function. These structures include bulges and internal loops that can form tertiary contacts or serve as ligand binding sites. The most commonly exploited RNA drug target for small molecule intervention is the bacterial ribosome, more specifically the rRNA aminoacyl-tRNA site (rRNA A-site) which is a major target for the aminoglycoside class of antibiotics. The bacterial A-site is composed of a 1 x 1 nucleotide all-U internal loop and a 2 x 1 nucleotide all-A internal loop separated by a single GC base pair. Therefore, we probed the molecular recognition of a small library of four aminoglycosides for binding a 16384-member bacterial rRNA A-site-like internal loop library using two-dimensional combinatorial screening (2DCS). 2DCS is a microarray-based method that probes RNA and chemical spaces simultaneously. These studies sought to determine if aminoglycosides select their therapeutic target if given a choice of binding all possible internal loops derived from an A-site-like library. Results show that the bacterial rRNA A-site was not selected by any aminoglycoside. Analyses of selected sequences using the RNA Privileged Space Predictor (RNA-PSP) program show that each aminoglycoside preferentially binds different types of internal loops. For three of the aminoglycosides, 6''-azido-kanamycin A, 5-O-(2-azidoethyl)-neamine, and 6''-azido-tobramycin, the selected internal loops bind with approximately 10-fold higher affinity than the bacterial rRNA A-site. The internal loops selected to bind 5''-azido-neomycin B bind with an affinity similar to that of the therapeutic target. Selected internal loops that are unique for each aminoglycoside have dissociation constants ranging from 25 to 270 nM and are specific for the aminoglycoside they was selected to bind compared to the other arrayed aminoglycosides. These studies further establish a database of RNA motifs that are recognized by small molecules that

  2. The HIV-1 Envelope Transmembrane Domain Binds TLR2 through a Distinct Dimerization Motif and Inhibits TLR2-Mediated Responses

    PubMed Central

    Rotem, Etai; Schwarzter, Roland; Gramatica, Andrea; Futerman, Anthony H.; Shai, Yechiel

    2014-01-01

    HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation. PMID:25121610

  3. The HIV-1 envelope transmembrane domain binds TLR2 through a distinct dimerization motif and inhibits TLR2-mediated responses.

    PubMed

    Reuven, Eliran Moshe; Ali, Mohammad; Rotem, Etai; Schwarzer, Roland; Schwarzter, Roland; Gramatica, Andrea; Futerman, Anthony H; Shai, Yechiel

    2014-08-01

    HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

  4. The splenic autoimmune response to ADAMTS13 in thrombotic thrombocytopenic purpura contains recurrent antigen-binding CDR3 motifs.

    PubMed

    Schaller, Monica; Vogel, Monique; Kentouche, Karim; Lämmle, Bernhard; Kremer Hovinga, Johanna A

    2014-11-27

    Acquired thrombotic thrombocytopenic purpura (TTP) is the consequence of a severe ADAMTS13 deficiency resulting from autoantibodies inhibiting ADAMTS13 or accelerating its clearance. Despite the success of plasma exchange the risk of relapse is high. From 2 patients (A and B), splenectomized for recurrent episodes of acquired TTP, the splenic B-cell response against ADAMTS13 was characterized through generation of human monoclonal anti-ADAMTS13 autoantibodies (mAbs) by cloning an immunoglobulin G (IgG)4κ- and IgG4λ-Fab library using phage display technology and by Epstein-Barr virus transformation of switched memory B cells (CD19+/CD27+/IgG+). Sequence analysis of the anti-ADAMTS13 IgGs of both patients revealed that the VH gene use was limited in our patients to VH1-3 (55%), VH1-69 (17%), VH3-30 (7%), and VH4-28 (21%) and contained 8 unique and thus far not reported heavy-chain complementarity determining region 3 motifs, of which 4 were shared by the 2 patients. The discovery of several highly similar anti-ADAMTS13 autoantibodies in 2 unrelated TTP patients suggests that the autoimmune response is antigen driven, because the probability that such similar immunoglobulin rearrangements happen by chance is very low (< 10(-9)).

  5. Fibronectin-Degrading Activity of Trypanosoma cruzi Cysteine Proteinase Plays a Role in Host Cell Invasion

    PubMed Central

    Maeda, Fernando Yukio; Cortez, Cristian; Izidoro, Mario Augusto; Juliano, Luiz

    2014-01-01

    Trypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insect-stage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. Using T. cruzi CL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the major T. cruzi cysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-β1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase of T. cruzi metacyclic forms, through its fibronectin-degrading activity, is implicated in host cell invasion. PMID:25267835

  6. An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α

    PubMed Central

    Rojas, Margarito; Vasconcelos, Gabriel; Dever, Thomas E.

    2015-01-01

    Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective mechanism cells use during stress conditions. Following relief of the stress, the growth arrest and DNA damage-inducible protein GADD34 associates with the broadly acting serine/threonine protein phosphatase 1 (PP1) to dephosphorylate eIF2α. Whereas the PP1-binding motif on GADD34 has been defined, it remains to be determined how GADD34 directs PP1 to specifically dephosphorylate eIF2α. In this report, we map a novel eIF2α-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. This motif is characterized by the consensus sequence Rx[Gnl]x1–2Wxxx[Arlv]x[Dn][Rg]xRFxx[Rlvk][Ivc], where capital letters are preferred and x is any residue. Point mutations altering the eIF2α-binding motif impair the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, this eIF2α-docking motif is conserved among viral orthologs of GADD34, and is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2α dephosphorylation. Taken together, these data indicate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs. PMID:26100893

  7. An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α.

    PubMed

    Rojas, Margarito; Vasconcelos, Gabriel; Dever, Thomas E

    2015-07-07

    Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective mechanism cells use during stress conditions. Following relief of the stress, the growth arrest and DNA damage-inducible protein GADD34 associates with the broadly acting serine/threonine protein phosphatase 1 (PP1) to dephosphorylate eIF2α. Whereas the PP1-binding motif on GADD34 has been defined, it remains to be determined how GADD34 directs PP1 to specifically dephosphorylate eIF2α. In this report, we map a novel eIF2α-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. This motif is characterized by the consensus sequence Rx[Gnl]x(1-2)Wxxx[Arlv]x[Dn][Rg]xRFxx[Rlvk][Ivc], where capital letters are preferred and x is any residue. Point mutations altering the eIF2α-binding motif impair the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, this eIF2α-docking motif is conserved among viral orthologs of GADD34, and is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2α dephosphorylation. Taken together, these data indicate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs.

  8. Purification, cloning, and expression of a murine phosphoprotein that binds the kappa B motif in vitro identifies it as the homolog of the human heterogeneous nuclear ribonucleoprotein K protein. Description of a novel DNA-dependent phosphorylation process.

    PubMed

    Ostrowski, J; Van Seuningen, I; Seger, R; Rauch, C T; Sleath, P R; McMullen, B A; Bomsztyk, K

    1994-07-01

    The kappa B enhancer element regulates expression of many genes involved in immune responses and other processes. kappa B motif binds a number of proteins, some but not all, are related to the NF-kappa B family of transcription factors. We have previously identified a 65-kDa phosphoprotein that is specifically recognized by the kappa B motif (Ostrowski, J., Sims, J. E., Sibley, C. H., Valentine, M. A., Dower, S. K., Meier, K. E., and Bomsztyk, K. (1991) J. Biol. Chem. 266, 12722-12733). This protein is closely associated with a serine/threonine kinase that is responsive to treatment of cells with interleukin-1 and other agents. We report here purification, cloning, and expression of this kappa B motif-binding phosphoprotein. The primary structure deduced from the isolated murine cDNA, identifies the protein as the homolog of the human heterogeneous nuclear ribonucleoprotein K protein. Antipeptide antibodies and expression of the cloned cDNA in Escherichia coli, demonstrated that the K protein is the authentic phosphoprotein that binds the kappa B motif in vitro. We also demonstrate that the in vitro phosphorylation of the natural and the recombinant K proteins by the associated kinase is stimulated by the kappa B motif.

  9. Histatins: salivary peptides with copper(II)- and zinc(II)-binding motifs: perspectives for biomedical applications.

    PubMed

    Melino, Sonia; Santone, Celeste; Di Nardo, Paolo; Sarkar, Bibudhendra

    2014-02-01

    Natural antimicrobial peptides represent a primordial mechanism of immunity in both vertebrate and nonvertebrate organisms. Among them, histatins belong to a family of human salivary metal-binding peptides displaying potent antibacterial, antifungal and wound-healing activities. These properties, along with the ability of histatins to inhibit collagenases and cysteine proteases, have attracted much attention for their potential use in the treatment of several oral diseases. This review critically assesses the studies carried out to date in order to provide a comprehensive and systematic vision of the information accumulated so far. In particular, the relationship between metal-binding and peptide activity is extensively analysed. The review provides important clues for developing possible therapeutic applications of histatins and their synthetic peptide analogues by creating a set of necessary resource materials to support investigators and industries interested in exploiting their unique properties.

  10. The structure of Plasmodium vivax phosphatidylethanolamine-binding protein suggests a functional motif containing a left-handed helix

    PubMed Central

    Arakaki, Tracy; Neely, Helen; Boni, Erica; Mueller, Natasha; Buckner, Frederick S.; Van Voorhis, Wesley C.; Lauricella, Angela; DeTitta, George; Luft, Joseph; Hol, Wim G. J.; Merritt, Ethan A.

    2007-01-01

    The structure of a putative Raf kinase inhibitor protein (RKIP) homolog from the eukaryotic parasite Plasmodium vivax has been studied to a resolution of 1.3 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protozoan protein is topologically similar to previously studied members of the phosphatidylethanolamine-binding protein (PEBP) sequence family, but exhibits a distinctive left-handed α-helical region at one side of the canonical phospholipid-binding site. Re-examination of previously determined PEBP structures suggests that the P. vivax protein and yeast carboxypeptidase Y inhibitor may represent a structurally distinct subfamily of the diverse PEBP-sequence family. PMID:17329808

  11. Osteopontin is cleaved at multiple sites close to its integrin-binding motifs in milk and is a novel substrate for plasmin and cathepsin D.

    PubMed

    Christensen, Brian; Schack, Lotte; Kläning, Eva; Sørensen, Esben S

    2010-03-12

    Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg-Gly-Asp(145) sequence and were generated by cleavage of the Leu(151)-Arg(152), Arg(152)-Ser(153), Ser(153)-Lys(154), Lys(154)-Ser(155), Ser(155)-Lys(156), Lys(156)-Lys(157), or Phe(158)-Arg(159) peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg(152)-Ser(153) matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys(154)-Ser(155). Another endogenous milk protease, cathepsin D, cleaved the Leu(151)-Arg(152) bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the alpha(V)beta(3)- or alpha(5)beta(1)-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.

  12. OsCCD1, a novel small calcium-binding protein with one EF-hand motif, positively regulates osmotic and salt tolerance in rice.

    PubMed

    Jing, Pei; Zou, Juanzi; Kong, Lin; Hu, Shiqi; Wang, Biying; Yang, Jun; Xie, Guosheng

    2016-06-01

    Calcium-binding proteins play key roles in the signal transduction in the growth and stress response in eukaryotes. However, a subfamily of proteins with one EF-hand motif has not been fully studied in higher plants. Here, a novel small calcium-binding protein with a C-terminal centrin-like domain (CCD1) in rice, OsCCD1, was characterized to show high similarity with a TaCCD1 in wheat. As a result, OsCCD1 can bind Ca(2+) in the in vitro EMSA and the fluorescence staining calcium-binding assays. Transient expression of green fluorescent protein (GFP)-tagged OsCCD1 in rice protoplasts showed that OsCCD1 was localized in the nucleus and cytosol of rice cells. OsCCD1 transcript levels were transiently induced by osmotic stress and salt stress through the calcium-mediated ABA signal. The rice seedlings of T-DNA mutant lines showed significantly less tolerance to osmotic and salt stresses than wild type plants (p<0.01). Conversely, its overexpressors can significantly enhance the tolerance to osmotic and salt stresses than wild type plants (p<0.05). Semi-quantitative RT-PCR analysis revealed that, OsDREB2B, OsAPX1 and OsP5CS genes are involved in the rice tolerance to osmotic and salt stresses. In sum, OsCCD1 gene probably affects the DREB2B and its downstream genes to positively regulate osmotic and salt tolerance in rice seedlings.

  13. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

    PubMed

    Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A; Brown, Elizabeth E; Sanderson, Ralph D

    2016-01-22

    Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.

  14. Rice MEL2, the RNA recognition motif (RRM) protein, binds in vitro to meiosis-expressed genes containing U-rich RNA consensus sequences in the 3'-UTR.

    PubMed

    Miyazaki, Saori; Sato, Yutaka; Asano, Tomoya; Nagamura, Yoshiaki; Nonomura, Ken-Ichi

    2015-10-01

    Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.

  15. The serine protease motif of Pic mediates a dose-dependent mucolytic activity after binding to sugar constituents of the mucin substrate.

    PubMed

    Gutiérrez-Jiménez, Javier; Arciniega, Ivonne; Navarro-García, Fernando

    2008-08-01

    The pic gene is harbored on the chromosomes of three important pathogens: enteroaggregative Escherichia coli (EAEC), uropathogenic E. coli (UPEC), and Shigella flexneri. Since Pic is secreted into the intestinal lumen during EAEC infection, we sought to identify intestinal-mucosal substrates for Pic. Pic did not damage epithelial cells, cleave fodrin, or degrade host defense proteins embedded in the mucus layer (sIgA, lactoferrin and lysozyme). However, by using a solid-phase assay to evaluate the mucinolytic activity of EAEC Pic, we documented a specific, dose-dependent mucinolytic activity. A serine protease inhibitor and an enzymatically inactive variant of Pic were used to show that the Pic serine protease motif is required for mucinolytic activity. Pic binds mucin, and this binding was blocked in competition assays using monosaccharide constituents of the oligosaccharide side chains of mucin. Moreover, Pic mucinolytic activity decreased when sialic acid was removed from mucin. Thus, Pic is a mucinase with lectin-like activity that can be related to its reported hemagglutinin activity. Our results suggest that EAEC may secrete Pic into the intestinal lumen as a strategy for penetrating the gel-like mucus layer during EAEC colonization.

  16. A Synthetic Peptide with the Putative Iron Binding Motif of Amyloid Precursor Protein (APP) Does Not Catalytically Oxidize Iron

    PubMed Central

    Ebrahimi, Kourosh Honarmand; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2012-01-01

    The β-amyloid precursor protein (APP), which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II) binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III) product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II) just as in ferritin. We measured the ferroxidase activity indirectly (i) by the incorporation of the Fe(III) product of the ferroxidase reaction into transferrin and directly (ii) by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II) oxidation by molecular oxygen. Zn(II) binds to transferrin and diminishes its Fe(III) incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and – by implication – of APP should be re-evaluated. PMID:22916096

  17. The involvement of an integrin-like protein and protein kinase C in amoebic adhesion to fibronectin and amoebic cytotoxicity.

    PubMed

    Han, Kyu-Lee; Lee, Hyun-Ju; Shin, Myeong Heon; Shin, Ho-Joon; Im, Kyung-Il; Park, Soon-Jung

    2004-09-01

    Adherence of a pathogen to the host cell is one of the critical steps in microbial infections. Naegleria fowleri, a causative agent of primary amoebic meningoencephalitis in humans, is expected to interact with extracellular components of the host, such as fibronectin, in a receptor-mediated mode. In this study, we investigated the interaction between N. fowleri and fibronectin to understand its cytopathology. In binding assays using immobilized fibronectin, the number of amoebae bound to fibronectin was increased compared to the controls, and was dependent on the amount of coated fibronectin present. A fibronectin binding protein of 60 kDa was found in extracts of N. fowleri. Western blot and immunolocalization assays using integrin alpha(5)/FnR antibodies showed that a 60 kDa protein reacted with the antibodies in extracts of N. fowleri, which was localized on the surface of N. fowleri. Preincubation of N. fowleri with the integrin antibodies significantly inhibited amoebic binding to fibronectin and cytotoxicity to the CHO cells. Additionally, protein kinase C activity was detected in the extract of N. fowleri. When N. fowleri was pretreated with protein kinase C activator or inhibitor, the abilities of amoebic adhesion to fibronectin and cytotoxicity to the host cells were markedly affected compared to untreated amoebae. These results suggest that an amoebic integrin-like receptor and protein kinase C play important roles in amoebic cellular processes in response to fibronectin.

  18. Candidate disease resistance genes in sunflower cloned using conserved nucleotide-binding site motifs: genetic mapping and linkage to the downy mildew resistance gene Pl1.

    PubMed

    Gedil, M A; Slabaugh, M B; Berry, S; Johnson, R; Michelmore, R; Miller, J; Gulya, T; Knapp, S J

    2001-04-01

    Disease resistance gene candidates (RGCs) belonging to the nucleotide-binding site (NBS) superfamily have been cloned from numerous crop plants using highly conserved DNA sequence motifs. The aims of this research were to (i) isolate genomic DNA clones for RGCs in cultivated sunflower (Helianthus annuus L.) and (ii) map RGC markers and Pl1, a gene for resistance to downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni) race 1. Degenerate oligonucleotide primers targeted to conserved NBS DNA sequence motifs were used to amplify RGC fragments from sunflower genomic DNA. PCR products were cloned, sequenced, and assigned to 11 groups. RFLP analyses mapped six RGC loci to three linkage groups. One of the RGCs (Ha-4W2) was linked to Pl1, a downy mildew resistance gene. A cleaved amplified polymorphic sequence (CAPS) marker was developed for Ha-4W2 using gene-specific oligonucleotide primers. Downy mildew susceptible lines (HA89 and HA372) lacked a 276-bp Tsp5091 restriction fragment that was present in downy mildew resistant lines (HA370, 335, 336, 337, 338, and 339). HA370 x HA372 F2 progeny were genotyped for the Ha-4W2 CAPS marker and phenotyped for resistance to downy mildew race 1. The CAPS marker was linked to but did not completely cosegregate with Pl1 on linkage group 8. Ha-4W2 was found to comprise a gene family with at least five members. Although genetic markers for Ha-4W2 have utility for marker-assisted selection, the RGC detected by the CAPS marker has been ruled out as a candidate gene for Pl1. Three of the RGC probes were monomorphic between HA370 and HA372 and still need to be mapped and screened for linkage to disease resistance loci.

  19. Motif enrichment tool.

    PubMed

    Blatti, Charles; Sinha, Saurabh

    2014-07-01

    The Motif Enrichment Tool (MET) provides an online interface that enables users to find major transcriptional regulators of their gene sets of interest. MET searches the appropriate regulatory region around each gene and identifies which transcription factor DNA-binding specificities (motifs) are statistically overrepresented. Motif enrichment analysis is currently available for many metazoan species including human, mouse, fruit fly, planaria and flowering plants. MET also leverages high-throughput experimental data such as ChIP-seq and DNase-seq from ENCODE and ModENCODE to identify the regulatory targets of a transcription factor with greater precision. The results from MET are produced in real time and are linked to a genome browser for easy follow-up analysis. Use of the web tool is free and open to all, and there is no login requirement. ADDRESS: http://veda.cs.uiuc.edu/MET/.

  20. PP1 phosphatase-binding motif in Reg1 protein of Saccharomyces cerevisiae is required for interaction with both the PP1 phosphatase Glc7 and the Snf1 protein kinase

    PubMed Central

    Tabba, Shadi; Mangat, Simmanjeet; McCartney, Rhonda; Schmidt, Martin C.

    2010-01-01

    In Saccharomyces cerevisiae, Snf1 kinase, the ortholog of the mammalian AMP-activated protein kinase, is activated by an increase in the phosphorylation of the conserved threonine residue in its activation loop. The phosphorylation status of this key site is determined by changes in the rate of dephosphorylation catalyzed by the yeast PP1 phosphatase Glc7 in a complex with the Reg1 protein. Reg1 and many PP1 phosphatase regulatory subunits utilize some variation of the conserved RVxF motif for interaction with PP1. In the Snf1 pathway, the exact role of the Reg1 protein is uncertain since it binds to both the Glc7 phosphatase and to Snf1, the Glc7 substrate. In this study we sought to clarify the role of Reg1 by separating the Snf1- and Glc7-binding functions. We generated a series of Reg1 proteins, some with deletions of conserved domains and one with two amino acid changes in the RVxF motif. The ability of Reg1 to bind Snf1 and Glc7 required the same domains of Reg1. Further, the RVxF motif that is essential for Reg1 binding to Glc7 is also required for binding to Snf1. Our data suggest that the regulation of Snf1 dephosphorylation is imparted through a dynamic competition between the Glc7 phosphatase and the Snf1 kinase for binding to the PP1 regulatory subunit Reg1. PMID:20170726

  1. Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

    SciTech Connect

    Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo; Lersch,Robert A.; Gee, Sherry L.; Hou, Victor C.; Lo, Annie J.; Short, Sarah A.; Chasis, Joel A.; Winkelmann, John C.; Conboy, John G.

    2006-03-01

    Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.

  2. Drosophila melanogaster Hox Transcription Factors Access the RNA Polymerase II Machinery through Direct Homeodomain Binding to a Conserved Motif of Mediator Subunit Med19

    PubM