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Sample records for adhesin complex protein

  1. Trimeric Autotransporter Adhesins in Members of the Burkholderia Cepacia Complex: A Multifunctional Family of Proteins Implicated in Virulence

    PubMed Central

    Mil-Homens, Dalila; Fialho, Arsénio M.

    2011-01-01

    Trimeric autotransporter adhesins (TAAs) are multimeric surface proteins exclusively found in bacteria. They are involved in various biological traits of pathogenic Gram-negative bacteria including adherence, biofilm formation, invasion, survival within eukaryotic cells, serum resistance, and cytotoxicity. TAAs have a modular architecture composed by a conserved membrane-anchored C-terminal domain and a variable number of stalk and head domains. In this study, a bioinformatic approach has been used to analyze the distribution and architecture of TAAs among Burkholderia cepacia complex (Bcc) genomes. Fifteen genomes were probed revealing a total of 74 encoding sequences. Compared with other bacterial species, the Bcc genomes contain a large number of TAAs (two genes to up to eight genes, such as in B. cenocepacia). Phylogenetic analysis showed that the TAAs grouped into at least eight distinct clusters. TAAs with serine-rich repeats are clearly well separated from others, thereby representing a different evolutionary lineage. Comparative gene mapping across Bcc genomes reveals that TAA genes are inserted within conserved synteny blocks. We further focused our analysis on the epidemic strain B. cenocepacia J2315 in which seven TAAs were annotated. Among these, three TAA-encoding genes (BCAM019, BCAM0223, and BCAM0224) are organized into a cluster and are candidates for multifunctional virulence factors. Here we review the current insights into the functional role of BCAM0224 as a model locus. PMID:22919579

  2. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  3. Autocatalytic association of proteins by covalent bond formation: a Bio Molecular Welding toolbox derived from a bacterial adhesin

    PubMed Central

    Bonnet, J.; Cartannaz, J.; Tourcier, G.; Contreras-Martel, C.; Kleman, J. P.; Morlot, C.; Vernet, T.; Di Guilmi, A. M.

    2017-01-01

    Unusual intramolecular cross-links present in adhesins from Gram-positive bacteria have been used to develop a generic process amenable to biotechnology applications. Based on the crystal structure of RrgA, the Streptococcus pneumoniae pilus adhesin, we provide evidence that two engineered protein fragments retain their ability to associate covalently with high specificity, in vivo and in vitro, once isolated from the parent protein. We determined the optimal conditions for the assembly of the complex and we solved its crystal structure at 2 Å. Furthermore, we demonstrate biotechnological applications related to antibody production, nanoassembly and cell-surface labeling based on this process we named Bio Molecular Welding. PMID:28252635

  4. 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

  5. The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation

    PubMed Central

    Diaz, Suraya A.; Martin, Stephen R.; Howell, Steven A.; Grainger, Munira; Moon, Robert W.; Green, Judith L.

    2016-01-01

    Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity. PMID

  6. The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation.

    PubMed

    Diaz, Suraya A; Martin, Stephen R; Howell, Steven A; Grainger, Munira; Moon, Robert W; Green, Judith L; Holder, Anthony A

    2016-01-01

    Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity.

  7. Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase

    PubMed Central

    Beaussart, Audrey; Brandhorst, Tristan

    2015-01-01

    ABSTRACT Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. PMID:26396244

  8. Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity

    PubMed Central

    Guo, Shuaiqi; Garnham, Christopher P.; Whitney, John C.; Graham, Laurie A.; Davies, Peter L.

    2012-01-01

    A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice. PMID:23144980

  9. The apicomplexan glideosome and adhesins -- structures and function

    PubMed Central

    Boucher, Lauren E.; Bosch, Jürgen

    2015-01-01

    The apicomplexan family of pathogens, which includes Plasmodium spp. and Toxoplasma gondii, are primarily obligate intracellular parasites and invade multiple cell types. These parasites express extracellular membrane protein receptors, adhesins, to form specific pathogen-host cell interaction complexes. Various adhesins are used to invade a variety of cell types. The receptors are linked to an actomyosin motor, which is part of a complex comprised of many proteins known as the invasion machinery or glideosome. To date, reviews on invasion have focused primarily on the molecular pathways and signals of invasion, with little or no structural information presented. Over 75 structures of parasite receptors and glideosome proteins have been deposited with the Protein Data Bank. These structures include adhesins, motor proteins, bridging proteins, inner membrane complex and cytoskeletal proteins, as well as co-crystal structures with peptides and antibodies. These structures provide information regarding key interactions necessary for target receptor engagement, machinery complex formation, how force is transmitted, and the basis of inhibitory antibodies. Additionally, these structures can provide starting points for the development of antibodies and inhibitory molecules targeting protein-protein interactions, with the aim to inhibit invasion. This review provides an overview of the parasite adhesin protein families, the glideosome components, glideosome architecture, and discuss recent work regarding alternative models. PMID:25764948

  10. Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli.

    PubMed

    Luna-Pineda, Víctor M; Reyes-Grajeda, Juan Pablo; Cruz-Córdova, Ariadnna; Saldaña-Ahuactzi, Zeus; Ochoa, Sara A; Maldonado-Bernal, Carmen; Cázares-Domínguez, Vicenta; Moreno-Fierros, Leticia; Arellano-Galindo, José; Hernández-Castro, Rigoberto; Xicohtencatl-Cortes, Juan

    2016-01-01

    Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA), and trimeric (fimH-csgA-papG) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa, corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. FimH, CsgA, and PapG stimulated the release of 372-398 pg/mL IL-6; interestingly, FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018) and 521.24 pg/mL (p ≤ 0.002) IL-6, respectively. In addition, FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001) and 450.40 pg/mL (p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit polyclonal antibodies

  11. Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli

    PubMed Central

    Luna-Pineda, Víctor M.; Reyes-Grajeda, Juan Pablo; Cruz-Córdova, Ariadnna; Saldaña-Ahuactzi, Zeus; Ochoa, Sara A.; Maldonado-Bernal, Carmen; Cázares-Domínguez, Vicenta; Moreno-Fierros, Leticia; Arellano-Galindo, José; Hernández-Castro, Rigoberto; Xicohtencatl-Cortes, Juan

    2016-01-01

    Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA), and trimeric (fimH-csgA-papG) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa, corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. FimH, CsgA, and PapG stimulated the release of 372–398 pg/mL IL-6; interestingly, FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018) and 521.24 pg/mL (p ≤ 0.002) IL-6, respectively. In addition, FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001) and 450.40 pg/mL (p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit polyclonal antibodies

  12. Spiroplasma eriocheiris Adhesin-Like Protein (ALP) Interacts with Epidermal Growth Factor (EGF) Domain Proteins to Facilitate Infection

    PubMed Central

    Hou, Libo; Liu, Yuhan; Gao, Qi; Xu, Xuechuan; Ning, Mingxiao; Bi, Jingxiu; Liu, Hui; Liu, Min; Gu, Wei; Wang, Wen; Meng, Qingguo

    2017-01-01

    Spiroplasma eriocheiris is a novel pathogen found in recent years, causing the tremor disease (TD) of Chinese mitten crab Eriocheir sinensis. Like Spiroplasma mirum, S. eriocheiris infects the newborn mouse (adult mice are not infected) and can cause cataract. Adhesion-related protein is an important protein involved in the interaction between pathogen and host. In this study, the Adhesin-like Protein (ALP) of S. eriocheiris was detected on its outer membrane by using immune electron microscopy, and was found to be involved in the bacterium's infection of mouse embryo fibroblasts (3T6-Swiss albino). Yeast two-hybrid analysis demonstrated that ALP interacts with a diverse group of mouse proteins. The interactions between recombinant partial fibulin7 (FBLN7; including two epidermal growth factor [EGF] domains) and ALP were confirmed by Far-western blotting and colocalization. We synthetized the domains of FBLN7 [EGF domain: amino acids 136–172 and complement control protein (CCP) domain: 81–134 amino acids], and demonstrated that only EGF domain of FBLN7 can interact with ALP. Because the EGF domain has high degree of similarity to EGF, it can activate the downstream EGFR signaling pathway, in key site amino acids. The EGFR pathway in 3T6 cells was restrained after rALP stimulation resulting from competitive binding of ALP to EGF. The unborn mouse, newborn mouse, and the adult mouse with cataract have a small amount of expressed FBLN7; however, none was detected in the brain and very little expression was seen in the eye of normal adult mice. In short, ALP as a S. eriocheiris surface protein, is critical for infection and further supports the role of ALP in S. eriocheiris infection by competitive effection of the EGF/EGFR axis of the target cells. PMID:28184355

  13. Cluster analysis identifies aminoacid compositional features that indicate Toxoplasma gondii adhesin proteins

    PubMed Central

    Arenas, Ailan F; Salcedo, Gladys E; Moncada, Diego M; Erazo, Diego A; Osorio, Juan F; Gomez-Marin, Jorge E

    2012-01-01

    Toxoplasma gondii invade host cells using a multi-step process that depends on the regulated secretion of adhesions. To identify key primary sequence features of adhesins in this parasite, we analyze the relative frequency of individual amino acids, their dipeptide frequencies, and the polarity, polarizability and Van der Waals volume of the individual amino acids by using cluster analysis. This method identified cysteine as a key amino acid in the Toxoplasma adhesin group. The best vector algorithm of non-concatenated features was for 2 attributes: the single amino acid relative frequency and the dipeptide frequency. Polarity, polarizability and Van der Waals volume were not good classificatory attributes. Single amino acid attributes clustered unambiguously 67 apicomplexan hypothetical adhesins. This algorithm was also useful for clustering hypothetical Toxoplasma target host receptors. All of the cluster performances had over 70% sensitivity and 80% specificity. Compositional aminoacid data can be useful for improving machine learning-based prediction software when homology and structural data are not sufficient. PMID:23144551

  14. Cohesive Properties of the Caulobacter crescentus Holdfast Adhesin Are Regulated by a Novel c-di-GMP Effector Protein

    PubMed Central

    Sprecher, Kathrin S.; Hug, Isabelle; Nesper, Jutta; Potthoff, Eva; Mahi, Mohamed-Ali; Sangermani, Matteo; Kaever, Volkhard; Schwede, Torsten; Vorholt, Julia

    2017-01-01

    ABSTRACT When encountering surfaces, many bacteria produce adhesins to facilitate their initial attachment and to irreversibly glue themselves to the solid substrate. A central molecule regulating the processes of this motile-sessile transition is the second messenger c-di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different bacterial model organisms. In Caulobacter crescentus, c-di-GMP regulates the synthesis of the polar holdfast adhesin during the cell cycle, yet the molecular and cellular details of this control are currently unknown. Here we identify HfsK, a member of a versatile N-acetyltransferase family, as a novel c-di-GMP effector involved in holdfast biogenesis. Cells lacking HfsK form highly malleable holdfast structures with reduced adhesive strength that cannot support surface colonization. We present indirect evidence that HfsK modifies the polysaccharide component of holdfast to buttress its cohesive properties. HfsK is a soluble protein but associates with the cell membrane during most of the cell cycle. Coincident with peak c-di-GMP levels during the C. crescentus cell cycle, HfsK relocalizes to the cytosol in a c-di-GMP-dependent manner. Our results indicate that this c-di-GMP-mediated dynamic positioning controls HfsK activity, leading to its inactivation at high c-di-GMP levels. A short C-terminal extension is essential for the membrane association, c-di-GMP binding, and activity of HfsK. We propose a model in which c-di-GMP binding leads to the dispersal and inactivation of HfsK as part of holdfast biogenesis progression. PMID:28325767

  15. Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica.

    PubMed Central

    Zhang, T; Stanley, S L

    1994-01-01

    The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with virulent E. histolytica trophozoites. PMID:8188384

  16. A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development

    PubMed Central

    Liu, Ming-Che; Yang, Cheng-Shou; Wang, Co-Shine

    2014-01-01

    The anther-specific gene LLA1271 isolated from lily (Lilium longiflorum Thunb.) anthers is novel and exists in two forms. The protein encoded by LLA1271 may represent an adhesin-like protein first found in higher plants. The protein contains a typical N-terminal signal peptide followed by a highly conserved repeat domain. The LLA1271 gene is temporally expressed at the phase of microspore development. RNA blot and RNA in situ hybridization analyses demonstrated that the gene was expressed both in the tapetum and in the microspore. The gene is endo- and exogenously induced by gibberellin. Studies with the gibberellin biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that LLA1271 is negatively regulated by ethylene, and a cross-talk of regulation between gibberellin and ethylene occurs in young anthers. The treatment with NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development in a state close to that of a tapetum without treatment. The LLA1271 protein is heat stable and heterogeneous. An immunoblot of separated protein fractions of the anther revealed that the LLA1271 protein was detected in protein fraction of the microspore released from the cell wall by treatment with either 0.5% or 2% Triton X-100. Ectopic expression of LLA1271 resulted in impaired stamen and low pollen germination. Scanning electron microscopy of TAP::LLA1271 pollen showed distorted exine formation and patterning. The LLA1271 protein once synthesized in both the tapetum and microspore is secreted and deposited on the surface of microspores, moderately affecting exine formation and patterning. PMID:24591055

  17. The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin

    PubMed Central

    2009-01-01

    Background The pathogenic fungus Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM). This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival. Results Here, we provide evidence that the malate synthase of P. brasiliensis (PbMLS) is located on the fungal cell surface, and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody was obtained against this protein. By using Confocal Laser Scanning Microscopy, PbMLS was detected in the cytoplasm and in the cell wall of the mother, but mainly of budding cells of the P. brasiliensis yeast phase. PbMLSr and its respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis with in vitro cultured epithelial cells A549. Conclusion These observations indicated that cell wall-associated PbMLS could be mediating the binding of fungal cells to the host, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection, behaving as an anchorless adhesin. PMID:20034376

  18. Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic.

    PubMed

    Binsker, Ulrike; Kohler, Thomas P; Krauel, Krystin; Kohler, Sylvia; Habermeyer, Johanna; Schwertz, Hansjörg; Hammerschmidt, Sven

    2017-04-07

    Streptococcus pneumoniae serotype 3 strains emerge frequently within clinical isolates of invasive diseases. Bacterial invasion into deeper tissues is associated with colonization and immune evasion mechanisms. Thus, pneumococci express a versatile repertoire of surface proteins sequestering and interacting specifically with components of the human extracellular matrix and serum. Hic, a PspC-like pneumococcal surface protein, possesses vitronectin and factor H binding activity. Here, we show that heterologously expressed Hic domains interact, similar to the classical PspC molecule, with human matricellular thrombospondin-1 (hTSP-1). Binding studies with isolated human thrombospondin-1 and various Hic domains suggest that the interaction between hTSP-1 and Hic differs from binding to vitronectin and factor H. Binding of Hic to hTSP-1 is inhibited by heparin and chondroitin sulfate A, indicating binding to the N-terminal globular domain or type I repeats of hTSP-1. Competitive inhibition experiments with other pneumococcal hTSP-1 adhesins demonstrated that PspC and PspC-like Hic recognize similar domains, whereas PavB and Hic can bind simultaneously to hTSP-1. In conclusion, Hic binds specifically hTSP-1; however, truncation in the N-terminal part of Hic decreases the binding activity, suggesting that the full length of the α-helical regions of Hic is required for an optimal interaction.

  19. The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins.

    PubMed

    Leo, Jack C; Lyskowski, Andrzej; Hattula, Katarina; Hartmann, Marcus D; Schwarz, Heinz; Butcher, Sarah J; Linke, Dirk; Lupas, Andrei N; Goldman, Adrian

    2011-07-13

    The Escherichia coli Ig-binding (Eib) proteins are trimeric autotransporter adhesins (TAAs) and receptors for IgG Fc. We present the structure of a large fragment of the passenger domain of EibD, the first TAA structure to have both a YadA-like head domain and the entire coiled-coil stalk. The stalk begins as a right-handed superhelix, but switches handedness halfway down. An unexpected β-minidomain joins the two and inserts a ∼120° rotation such that there is no net twist between the beginning and end of the stalk. This may be important in folding and autotransport. The surprisingly large cavities we found in EibD and other TAAs may explain how TAAs bend to bind their ligands. We identified how IgA and IgG bind and modeled the EibD-IgG Fc complex. We further show that EibD promotes autoagglutination and biofilm formation and forms a fibrillar layer covering the cell surface making zipper-like contacts between cells.

  20. Structural mechanisms underlying sequence-dependent variations in GAG affinities of decorin binding protein A, a Borrelia burgdorferi adhesin.

    PubMed

    Morgan, Ashli M; Wang, Xu

    2015-05-01

    Decorin-binding protein A (DBPA) is an important surface adhesin of the bacterium Borrelia burgdorferi, the causative agent of Lyme disease. DBPA facilitates the bacteria's colonization of human tissue by adhering to glycosaminoglycan (GAG), a sulfated polysaccharide. Interestingly, DBPA sequence variation among different strains of Borrelia spirochetes is high, resulting in significant differences in their GAG affinities. However, the structural mechanisms contributing to these differences are unknown. We determined the solution structures of DBPAs from strain N40 of B. burgdorferi and strain PBr of Borrelia garinii, two DBPA variants whose GAG affinities deviate significantly from strain B31, the best characterized version of DBPA. Our structures revealed that significant differences exist between PBr DBPA and B31/N40 DBPAs. In particular, the C-terminus of PBr DBPA, unlike C-termini from B31 and N40 DBPAs, is positioned away from the GAG-binding pocket and the linker between helices one and two of PBr DBPA is highly structured and retracted from the GAG-binding pocket. The repositioning of the C-terminus allowed the formation of an extra GAG-binding epitope in PBr DBPA and the retracted linker gave GAG ligands more access to the GAG-binding epitopes than other DBPAs. Characterization of GAG ligands' interactions with wild-type (WT) PBr and mutants confirmed the importance of the second major GAG-binding epitope and established the fact that the two epitopes are independent of one another and the new epitope is as important to GAG binding as the traditional epitope.

  1. The Bfp60 surface adhesin is an extracellular matrix and plasminogen protein interacting in Bacteroides fragilis

    PubMed Central

    de Oliveira Ferreira, Eliane; Teixeira, Felipe; Cordeiro, Fabiana; Lobo, Leandro Araujo; Rocha, Edson R.; Smith, Jeffrey C.; Domingues, Regina M C P

    2014-01-01

    Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis can not be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis. PMID:23850366

  2. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.

    PubMed

    Zakeri, Bijan; Fierer, Jacob O; Celik, Emrah; Chittock, Emily C; Schwarz-Linek, Ulrich; Moy, Vincent T; Howarth, Mark

    2012-03-20

    Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

  3. Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae

    PubMed Central

    Tavakoli, Minoo; Bouzari, Saeed; Siadat, Seyed Davar; Najar Peerayeh, Shahin; Jafari, Anis

    2015-01-01

    Background: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis. Objectives: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study. Materials and Methods: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting. Results: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration. Conclusions: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway. PMID:26034537

  4. Targeted protein engineering provides insights into binding mechanism and affinities of bacterial collagen adhesins.

    PubMed

    Ross, Caná L; Liang, Xiaowen; Liu, Qing; Murray, Barbara E; Höök, Magnus; Ganesh, Vannakambadi K

    2012-10-05

    The collagen-binding bacterial proteins, Ace and Cna, are well characterized on the biochemical and structural level. Despite overall structural similarity, recombinant forms of the Ace and Cna ligand-binding domains exhibit significantly different affinities and binding kinetics for collagen type I (CI) in vitro. In this study, we sought to understand, in submolecular detail, the bases for these differences. Using a structure-based approach, we engineered Cna and Ace variants by altering specific structural elements within the ligand-binding domains. Surface plasmon resonance-based binding analysis demonstrated that mutations that are predicted to alter the orientation of the Ace and Cna N(1) and N(2) subdomains significantly affect the interaction between the MSCRAMM (microbial surface components recognizing adhesive matrix molecule) and CI in vitro, including affinity, association/dissociation rates and binding ratio. Moreover, we utilized this information to engineer an Ace variant with an 11,000-fold higher CI affinity than the parent protein. Finally, we noted that several engineered proteins that exhibited a weak interaction with CI recognized more sites on CI, suggesting an inverse correlation between affinity and specificity.

  5. Protein E of Haemophilus influenzae is a ubiquitous highly conserved adhesin.

    PubMed

    Singh, Birendra; Brant, Marta; Kilian, Mogens; Hallström, Björn; Riesbeck, Kristian

    2010-02-01

    Protein E (PE) of nontypeable Haemophilus influenzae (NTHi) is involved in adhesion and activation of epithelial cells. A total of 186 clinical NTHi isolates, encapsulated H. influenzae, and culture collection strains were analyzed. PE was highly conserved in both NTHi and encapsulated H. influenzae (96.9%-100% identity without the signal peptide). PE also existed in other members of the genus Pasteurellaceae. The epithelial cell binding region (amino acids 84-108) was completely conserved. Phylogenetic analysis of the pe sequence separated Haemophilus species into 2 separate clusters. Importantly, PE was expressed in 98.4% of all NTHi (126 isolates) independently of the growth phase.

  6. Anaplasma marginale Outer Membrane Protein A Is an Adhesin That Recognizes Sialylated and Fucosylated Glycans and Functionally Depends on an Essential Binding Domain.

    PubMed

    Hebert, Kathryn S; Seidman, David; Oki, Aminat T; Izac, Jerilyn; Emani, Sarvani; Oliver, Lee D; Miller, Daniel P; Tegels, Brittney K; Kannagi, Reiji; Marconi, Richard T; Carlyon, Jason A

    2017-03-01

    Anaplasma marginale causes bovine anaplasmosis, a debilitating and potentially fatal tick-borne infection of cattle. Because A. marginale is an obligate intracellular organism, its adhesins that mediate entry into host cells are essential for survival. Here, we demonstrate that A. marginale outer membrane protein A (AmOmpA; AM854) contributes to the invasion of mammalian and tick host cells. AmOmpA exhibits predicted structural homology to OmpA of A. phagocytophilum (ApOmpA), an adhesin that uses key lysine and glycine residues to interact with α2,3-sialylated and α1,3-fucosylated glycan receptors, including 6-sulfo-sialyl Lewis x (6-sulfo-sLe(x)). Antisera against AmOmpA or its predicted binding domain inhibits A. marginale infection of host cells. Residues G55 and K58 are contributory, and K59 is essential for recombinant AmOmpA to bind to host cells. Enzymatic removal of α2,3-sialic acid and α1,3-fucose residues from host cell surfaces makes them less supportive of AmOmpA binding. AmOmpA is both an adhesin and an invasin, as coating inert beads with it confers adhesiveness and invasiveness. Recombinant forms of AmOmpA and ApOmpA competitively antagonize A. marginale infection of host cells, but a monoclonal antibody against 6-sulfo-sLe(x) fails to inhibit AmOmpA adhesion and A. marginale infection. Thus, the two OmpA proteins bind related but structurally distinct receptors. This study provides a detailed understanding of AmOmpA function, identifies its essential residues that can be targeted by blocking antibody to reduce infection, and determines that it binds to one or more α2,3-sialylated and α1,3-fucosylated glycan receptors that are unique from those targeted by ApOmpA.

  7. Piracy of adhesins: attachment of superinfecting pathogens to respiratory cilia by secreted adhesins of Bordetella pertussis.

    PubMed

    Tuomanen, E

    1986-12-01

    Two proteins secreted by Bordetella pertussis are known to mediate adherence of these bacteria to mammalian respiratory cilia. When either ciliated cells or other pathogenic bacteria were pretreated with these adhesins, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus acquired the ability to adhere to cilia in vitro and in vivo. Such piracy of adhesins may contribute to superinfection in mucosal diseases such as whooping cough.

  8. Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein.

    PubMed Central

    Hanson, M S; Hempel, J; Brinton, C C

    1988-01-01

    Type 1 pili of Escherichia coli contain three integral minor proteins with apparent molecular weights (Mr) of 28,000 (28K protein), 16,500, and 14,500 attached to rods composed of Mr-17,000 pilin subunits (Hanson and Brinton, Nature [London] 322:265-268). We describe here an improvement on our earlier method of pilus purification, which gives higher yields and higher purity. Also reported are methods allowing fractionation of intact type 1 pili into rods of pure pilin and free minor proteins, as well as fractionation of the 28K tip adhesion protein from the 16.5K and 14.5K proteins. We have determined the amino acid composition and amino-terminal sequence of the adhesion protein. This sequence shows limited homology with the amino-terminal sequences of several E. coli pilins, including type 1. Images PMID:2900235

  9. Vaccine efficacy of the attenuated Erysipelothrix rhusiopathiae YS-19 expressing a recombinant protein of Mycoplasma hyopneumoniae P97 adhesin against mycoplasmal pneumonia of swine.

    PubMed

    Shimoji, Yoshihiro; Oishi, Eiji; Muneta, Yoshihiro; Nosaka, Hideji; Mori, Yasuyuki

    2003-01-17

    The attenuated Erysipelothrix rhusiopathiae YS-19 strain was constructed for the purpose of delivering the C-terminal portion of the Mycoplasma hyopneumoniae P97 adhesin to the mucosal surface of the respiratory tract of pigs. In this study, the efficacy of the YS-19 vaccine against mycoplasmal pneumonia of swine was evaluated. Animal experiments revealed that intranasal immunization of pigs with the YS-19 strain significantly reduced the severity of pneumonic lung lesions caused by M. hyopneumoniae infection. In YS-19-immunized pigs, P97-specific serum antibodies were not detected. However, when stimulated with the P97 protein, peripheral blood mononuclear cells from the YS-19-immunized pigs had a significantly higher stimulation index (P<0.05) than that of cells from control pigs at 7 days post-challenge.

  10. Carbohydrate Receptors of Bacterial Adhesins: Implications and Reflections

    NASA Astrophysics Data System (ADS)

    Ohlsen, K.; Oelschlaeger, T. A.; Hacker, J.; Khan, A. S.

    Bacteria entering a host depend on adhesins to achieve colonization. Adhesins are bacterial surface structures mediating binding to host surficial areas. Most adhesins are composed of one or several proteins. Usually a single bacterial strain is able to express various adhesins. The adhesion type expressed may influence host-, tissue or even cell tropism of Gram-negative and of Gram-positive bacteria. The binding of fimbrial as well as of afimbrial adhesins of Gram-negative bacteria to host carbohydrate structures (=receptors) has been elucidated in great detail. In contrast, in Gram-positives, most well studied adhesins bind to proteinaceous partners. Nevertheless, for both bacterial groups the binding of bacterial adhesins to eukaryotic carbohydrate receptors is essential for establishing colonization or infection. The characterization of this interaction down to the submolecular level provides the basis for strategies to interfere with this early step of infection which should lead to the prevention of subsequent disease. However, this goal will not be achieved easily because bacterial adherence is not a monocausal event but rather mediated by a variety of adhesins.

  11. Proteins : paradigms of complexity /

    SciTech Connect

    Frauenfelder, Hans,

    2001-01-01

    Proteins are the working machines of living systems. Directed by the DNA, of the order of a few hundred building blocks, selected from twenty different amino acids, are covalently linked into a linear polypeptide chain. In the proper environment, the chain folds into the working protein, often a globule of linear dimensions of a few nanometers. The biologist considers proteins units from which living systems are built. Many physical scientists look at them as systems in which the laws of complexity can be studied better than anywhere else. Some of the results of such studies will be sketched.

  12. Identification of glycoprotein receptors within the human salivary proteome for the lectin-like BabA and SabA adhesins of Helicobacter pylori by fluorescence-based 2-D bacterial overlay.

    PubMed

    Walz, Anke; Odenbreit, Stefan; Stühler, Kai; Wattenberg, Andreas; Meyer, Helmut E; Mahdavi, Jafar; Borén, Thomas; Ruhl, Stefan

    2009-03-01

    Because gastric infection by Helicobacter pylori takes place via the oral route, possible interactions of this bacterium with human salivary proteins could occur. By using modified 1- and 2-D bacterial overlay, binding of H. pylori adhesins BabA and SabA to the whole range of salivary proteins was explored. Bound salivary receptor molecules were identified by MALDI-MS and by comparison to previously established proteome maps of whole and glandular salivas. By use of adhesin-deficient mutants, binding of H. pylori to MUC7 and gp-340 could be linked to the SabA and BabA adhesins, respectively, whereas binding to MUC5B was associated with both adhesins. Binding of H. pylori to the proline-rich glycoprotein was newly detected and assigned to BabA adhesin whereas the SabA adhesin was found to mediate binding to newly detected receptor molecules, including carbonic anhydrase VI, secretory component, heavy chain of secretory IgA1, parotid secretory protein and zinc-alpha(2)-glycoprotein. Some of these salivary glycoproteins are known to act as scavenger molecules or are involved in innate immunity whereas others might come to modify the pathogenetic properties of this organism. In general, this 2-D bacterial overlay technique represents a useful supplement in adhesion studies of bacteria with complex protein mixtures.

  13. A high-molecular-weight outer membrane protein of Xanthomonas oryzae pv. oryzae exhibits similarity to non-fimbrial adhesins of animal pathogenic bacteria and is required for optimum virulence.

    PubMed

    Ray, Suvendra K; Rajeshwari, R; Sharma, Yogendra; Sonti, Ramesh V

    2002-11-01

    Transposon insertions in a novel 3.798 kb open reading frame (ORF) of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo) cause virulence deficiency and altered colony/lawn morphology. This ORF encodes a protein, XadA, of 1,265 amino acids that exhibits significant similarity to non-fimbrial adhesins of animal pathogenic bacteria such as Yersinia YadA and Moraxella UspA1. An interesting feature is that the YadA similarity region is repeated six times within the XadA sequence and encompasses almost the entire length of the protein. Anti-XadA antibodies identified a 110 kDa outer membrane protein that was sensitive to protease treatment of whole cells. XadA expression is induced in minimal medium. Homology modelling suggests that XadA adopts a beta-helix conformation-like pertactin, a non-fimbrial adhesin of Bordetella pertussis. This work is the first characterization of a non-fimbrial adhesin-like molecule in a plant pathogenic bacterium. It extends our knowledge about the repertoire of homologous virulence factors that are deployed by animal and plant pathogenic bacteria to include functions potentially involved in adhesion.

  14. The Fusobacterium nucleatum Outer Membrane Protein RadD Is an Arginine-Inhibitable Adhesin Required for Inter-Species Adherence and the Structured Architecture of Multi-Species Biofilm

    PubMed Central

    Kaplan, Christopher W.; Lux, Renate; Haake, Susan Kinder; Shi, Wenyuan

    2009-01-01

    Summary A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central “bridging organisms” in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum. Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD) demonstrated significantly decreased co-aggregation with representatives of the Gram-positive “early oral colonizers”. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation. PMID:19007407

  15. Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine

    PubMed Central

    Mizrachi Nebenzahl, Yaffa; Blau, Karin; Kushnir, Tatyana; Shagan, Marilou; Portnoi, Maxim; Cohen, Aviad; Azriel, Shalhevet; Malka, Itai; Adawi, Asad; Kafka, Daniel; Dotan, Shahar; Guterman, Gali; Troib, Shany; Fishilevich, Tali; Gershoni, Jonathan M; Braiman, Alex; Mitchell, Andrea M; Mitchell, Timothy J; Porat, Nurith; Goliand, Inna; Chalifa Caspi, Vered; Swiatlo, Edwin; Tal, Michael; Ellis, Ronald; Elia, Natalie; Dagan, Ron

    2016-01-01

    In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development. PMID:26990554

  16. Interaction of Candida albicans cell wall Als3 protein with Streptococcus gordonii SspB adhesin promotes development of mixed-species communities.

    PubMed

    Silverman, Richard J; Nobbs, Angela H; Vickerman, M Margaret; Barbour, Michele E; Jenkinson, Howard F

    2010-11-01

    Candida albicans colonizes human mucosa and prosthetic surfaces associated with artificial joints, catheters, and dentures. In the oral cavity, C. albicans coexists with numerous bacterial species, and evidence suggests that bacteria may modulate fungal growth and biofilm formation. Streptococcus gordonii is found on most oral cavity surfaces and interacts with C. albicans to promote hyphal and biofilm formation. In this study, we investigated the role of the hyphal-wall protein Als3p in interactions of C. albicans with S. gordonii. Utilizing an ALS3 deletion mutant strain, it was shown that cells were not affected in initial adherence to the salivary pellicle or in hyphal formation in the planktonic phase. However, the Als3(-) mutant was unable to form biofilms on the salivary pellicle or deposited S. gordonii DL1 wild-type cells, and after initial adherence, als3Δ/als3Δ (ΔALS3) cells became detached concomitant with hyphal formation. In coaggregation assays, S. gordonii cells attached to, and accumulated around, hyphae formed by C. albicans wild-type cells. However, streptococci failed to attach to hyphae produced by the ΔALS3 mutant. Saccharomyces cerevisiae S150-2B cells expressing Als3p, but not control cells, supported binding of S. gordonii DL1. However, S. gordonii Δ(sspA sspB) cells deficient in production of the surface protein adhesins SspA and SspB showed >50% reduced levels of binding to S. cerevisiae expressing Als3p. Lactococcus lactis cells expressing SspB bound avidly to S. cerevisiae expressing Als3p, but not to S150-2B wild-type cells. These results show that recognition of C. albicans by S. gordonii involves Als3 protein-SspB protein interaction, defining a novel mechanism in fungal-bacterial communication.

  17. Structures of the Toxoplasma gliding motility adhesin

    PubMed Central

    Song, Gaojie; Springer, Timothy A.

    2014-01-01

    Micronemal protein 2 (MIC2) is the key adhesin that supports gliding motility and host cell invasion by Toxoplasma gondii. With a von Willebrand factor A (VWA) domain and six thrombospondin repeat domains (TSR1–6) in its ectodomain, MIC2 connects to the parasite actomyosin system through its cytoplasmic tail. MIC2-associated protein (M2AP) binds noncovalently to the MIC2 ectodomain. MIC2 and M2AP are stored in micronemes as proforms. We find that the MIC2–M2AP ectodomain complex is a highly elongated 1:1 monomer with M2AP bound to the TSR6 domain. Crystal structures of N-terminal fragments containing the VWA and TSR1 domains for proMIC2 and MIC2 reveal a closed conformation of the VWA domain and how it associates with the TSR1 domain. A long, proline-rich, disulfide-bonded pigtail loop in TSR1 overlaps the VWA domain. Mannose α-C-linked to Trp-276 in TSR1 has an unusual 1C4 chair conformation. The MIC2 VWA domain includes a mobile α5-helix and a 22-residue disordered region containing two disulfide bonds in place of an α6-helix. A hydrophobic residue in the prodomain binds to a pocket adjacent to the α7-helix that pistons in opening of the VWA domain to a putative high-affinity state. PMID:24639528

  18. Lectin-Glycan Interaction Network-Based Identification of Host Receptors of Microbial Pathogenic Adhesins

    PubMed Central

    Ielasi, Francesco S.; Alioscha-Perez, Mitchel; Donohue, Dagmara; Claes, Sandra; Sahli, Hichem; Schols, Dominique

    2016-01-01

    ABSTRACT The first step in the infection of humans by microbial pathogens is their adherence to host tissue cells, which is frequently based on the binding of carbohydrate-binding proteins (lectin-like adhesins) to human cell receptors that expose glycans. In only a few cases have the human receptors of pathogenic adhesins been described. A novel strategy—based on the construction of a lectin-glycan interaction (LGI) network—to identify the potential human binding receptors for pathogenic adhesins with lectin activity was developed. The new approach is based on linking glycan array screening results of these adhesins to a human glycoprotein database via the construction of an LGI network. This strategy was used to detect human receptors for virulent Escherichia coli (FimH adhesin), and the fungal pathogens Candida albicans (Als1p and Als3p adhesins) and C. glabrata (Epa1, Epa6, and Epa7 adhesins), which cause candidiasis. This LGI network strategy allows the profiling of potential adhesin binding receptors in the host with prioritization, based on experimental binding data, of the most relevant interactions. New potential targets for the selected adhesins were predicted and experimentally confirmed. This methodology was also used to predict lectin interactions with envelope glycoproteins of human-pathogenic viruses. It was shown that this strategy was successful in revealing that the FimH adhesin has anti-HIV activity. PMID:27406561

  19. A multi-repeat adhesin of the phytopathogen, Pectobacterium atrosepticum, is secreted by a Type I pathway and is subject to complex regulation involving a non-canonical diguanylate cyclase.

    PubMed

    Pérez-Mendoza, Daniel; Coulthurst, Sarah J; Humphris, Sonia; Campbell, Emma; Welch, Martin; Toth, Ian K; Salmond, George P C

    2011-11-01

    Cyclic diguanylate (c-di-GMP) is a second messenger controlling many important bacterial processes. The phytopathogen Pectobacterium atrosepticum SCRI1043 (Pba1043) possesses a Type I secretion system (T1SS) essential for the secretion of a proteinaceous multi-repeat adhesin (MRP) required for binding to the host plant. The genes encoding the MRP and the T1SS are tightly linked to genes encoding several putative c-di-GMP regulatory components. We show that c-di-GMP regulates secreted MRP levels in Pba1043 through the action of two genes encoding predicted diguanylate cyclase (DGC) and phosphodiesterase proteins (ECA3270 and ECA3271). Phenotypic analyses and quantification of c-di-GMP levels demonstrated that ECA3270 and ECA3271 regulate secreted MRP levels by increasing and decreasing, respectively, the intracellular levels of c-di-GMP. Moreover, ECA3270 represents the first active DGC reported to have an alternative active-site motif from the 'canonical' GG[D/E]EF. ECA3270 has an A-site motif of SGDEF and analysis of single amino acid replacements demonstrated that the first position of this motif can tolerate functional substitution. Serine in position one of the A-site is also observed in many other DGCs. Finally, another T1SS-linked regulator (ECA3265) also plays an important role in regulating secreted MRP, with an altered localization of MRP observed in an ECA3265 mutant background. Mutants defective in these three T1SS-linked regulators exhibit a reduction in root binding and virulence, confirming that this complex, finely tuned regulation system is crucial in the interaction with host plants.

  20. Structural Context for Protein N-glycosylation in Bacteria: The Structure of PEB3, an Adhesin from Campylobacter Jejuni

    SciTech Connect

    Rangarajan,E.; Bhatia, S.; Watson, D.; Munger, C.; Cygler, M.; Matte, A.; Young, N.

    2007-01-01

    Campylobacter jejuni is unusual among bacteria in possessing a eukaryotic-like system for N-linked protein glycosylation at Asn residues in sequons of the type Asp/Glu-Xaa-Asn-Xaa-Ser/Thr. However, little is known about the structural context of the glycosylated sequons, limiting the design of novel recombinant glycoproteins. To obtain more information on sequon structure, we have determined the crystal structure of the PEB3 (Cj0289c) dimer. PEB3 has the class II periplasmic-binding protein fold, with each monomer having two domains with a ligand-binding site containing citrate located between them, and overall resembles molybdate- and sulfate-binding proteins. The sequon around Asn90 is located within a surface-exposed loop joining two structural elements. The three key residues are well exposed on the surface; hence, they may be accessible to the PglB oligosaccharyltransferase in the folded state.

  1. The Afa/Dr adhesins of diffusely adhering Escherichia coli stimulate interleukin-8 secretion, activate mitogen-activated protein kinases, and promote polymorphonuclear transepithelial migration in T84 polarized epithelial cells.

    PubMed

    Bétis, Fréderic; Brest, Patrick; Hofman, Véronique; Guignot, Julie; Bernet-Camard, Marie-Françoise; Rossi, Bernard; Servin, Alain; Hofman, Paul

    2003-03-01

    Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and the E. coli laboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody that recognized the short consensus repeat 3 domain of DAF (monoclonal antibody 1H4). Moreover, Afa/Dr DAEC strains induced tyrosine phosphorylation of several T84 proteins and activated the mitogen-activated protein kinases (ERK1/2 mitogen-activated protein, P38, and Jun-C kinases). These data demonstrated for the first time that, in vitro, Afa/Dr DAEC strains exert a proinflammatory signal in intestinal epithelial cells.

  2. 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

  3. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

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

    2005-05-01

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

  4. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  5. Identification of a Collagen Type I Adhesin of Bacteroides fragilis

    PubMed Central

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

    2014-01-01

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

  6. Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli.

    PubMed

    Keller, Rogéria; Ordoñez, Juana G; de Oliveira, Rosana R; Trabulsi, Luiz R; Baldwin, Thomas J; Knutton, Stuart

    2002-05-01

    O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.

  7. Identification and Characterization of an Escorter for Two Secretory Adhesins in Toxoplasma gondii

    PubMed Central

    Reiss, Matthias; Viebig, Nicola; Brecht, Susan; Fourmaux, Marie-Noelle; Soete, Martine; Di Cristina, Manlio; Dubremetz, Jean François; Soldati, Dominique

    2001-01-01

    The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite. PMID:11157983

  8. Identification of novel adhesins of M. tuberculosis H37Rv using integrated approach of multiple computational algorithms and experimental analysis.

    PubMed

    Kumar, Sanjiv; Puniya, Bhanwar Lal; Parween, Shahila; Nahar, Pradip; Ramachandran, Srinivasan

    2013-01-01

    Pathogenic bacteria interacting with eukaryotic host express adhesins on their surface. These adhesins aid in bacterial attachment to the host cell receptors during colonization. A few adhesins such as Heparin binding hemagglutinin adhesin (HBHA), Apa, Malate Synthase of M. tuberculosis have been identified using specific experimental interaction models based on the biological knowledge of the pathogen. In the present work, we carried out computational screening for adhesins of M. tuberculosis. We used an integrated computational approach using SPAAN for predicting adhesins, PSORTb, SubLoc and LocTree for extracellular localization, and BLAST for verifying non-similarity to human proteins. These steps are among the first of reverse vaccinology. Multiple claims and attacks from different algorithms were processed through argumentative approach. Additional filtration criteria included selection for proteins with low molecular weights and absence of literature reports. We examined binding potential of the selected proteins using an image based ELISA. The protein Rv2599 (membrane protein) binds to human fibronectin, laminin and collagen. Rv3717 (N-acetylmuramoyl-L-alanine amidase) and Rv0309 (L,D-transpeptidase) bind to fibronectin and laminin. We report Rv2599 (membrane protein), Rv0309 and Rv3717 as novel adhesins of M. tuberculosis H37Rv. Our results expand the number of known adhesins of M. tuberculosis and suggest their regulated expression in different stages.

  9. Localization and characterization of Xylella fastidiosa haemagglutinin adhesins.

    PubMed

    Voegel, Tanja M; Warren, Jeremy G; Matsumoto, Ayumi; Igo, Michele M; Kirkpatrick, Bruce C

    2010-07-01

    Xylella fastidiosa is a gram-negative, xylem-inhabiting, plant-pathogenic bacterium responsible for several important diseases including Pierce's disease (PD) of grapevines. The bacteria form biofilms in grapevine xylem that contribute to the occlusion of the xylem vessels. X. fastidiosa haemagglutinin (HA) proteins are large afimbrial adhesins that have been shown to be crucial for biofilm formation. Little is known about the mechanism of X. fastidiosa HA-mediated cell-cell aggregation or the localization of the adhesins on the cell. We generated anti-HA antibodies and show that X. fastidiosa HAs are present in the outer membrane and secreted both as soluble proteins and in membrane vesicles. Furthermore, the HA pre-proteins are processed from the predicted molecular mass of 360 kDa to a mature 220 kDa protein. Based on this information, we are evaluating a novel form of potential resistance against PD by generating HA-expressing transgenic grapevines.

  10. The Biology of Neisseria Adhesins

    PubMed Central

    Hung, Miao-Chiu; Christodoulides, Myron

    2013-01-01

    Members of the genus Neisseria include pathogens causing important human diseases such as meningitis, septicaemia, gonorrhoea and pelvic inflammatory disease syndrome. Neisseriae are found on the exposed epithelia of the upper respiratory tract and the urogenital tract. Colonisation of these exposed epithelia is dependent on a repertoire of diverse bacterial molecules, extending not only from the surface of the bacteria but also found within the outer membrane. During invasive disease, pathogenic Neisseriae also interact with immune effector cells, vascular endothelia and the meninges. Neisseria adhesion involves the interplay of these multiple surface factors and in this review we discuss the structure and function of these important molecules and the nature of the host cell receptors and mechanisms involved in their recognition. We also describe the current status for recently identified Neisseria adhesins. Understanding the biology of Neisseria adhesins has an impact not only on the development of new vaccines but also in revealing fundamental knowledge about human biology. PMID:24833056

  11. The Cation-Responsive Protein NhaR of Escherichia coli Activates pgaABCD Transcription, Required for Production of the Biofilm Adhesin Poly-β-1,6-N-Acetyl-d-Glucosamine▿

    PubMed Central

    Goller, Carlos; Wang, Xin; Itoh, Yoshikane; Romeo, Tony

    2006-01-01

    The pgaABCD operon of Escherichia coli is required for production of the biofilm adhesin poly-β-1,6-N-acetyl-d-glucosamine (PGA). We establish here that NhaR, a DNA-binding protein of the LysR family of transcriptional regulators, activates transcription of this operon. Disruption of the nhaR gene decreased biofilm formation without affecting planktonic growth. PGA production was undetectable in an nhaR mutant strain. Expression of a pgaA′-′lacZ translational fusion was induced by NaCl and alkaline pH, but not by CaCl2 or sucrose, in an nhaR-dependent fashion. Primer extension and quantitative real-time reverse transcription-PCR analyses further revealed that NhaR affects the steady-state level of pga mRNA. A purified recombinant NhaR protein bound specifically and with high affinity within the pgaABCD promoter region; one apparent binding site overlaps the −35 element, and a second site lies immediately upstream of the first. This protein was necessary and sufficient for activation of in vitro transcription from the pgaA promoter. These results define a novel mechanism for regulation of biofilm formation in response to environmental conditions and suggest an expanded role for NhaR in promoting bacterial survival. PMID:16997959

  12. Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy.

    PubMed

    Arnal, L; Longo, G; Stupar, P; Castez, M F; Cattelan, N; Salvarezza, R C; Yantorno, O M; Kasas, S; Vela, M E

    2015-11-07

    Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.

  13. Lipoprotein CD0873 is a novel adhesin of Clostridium difficile.

    PubMed

    Kovacs-Simon, Andrea; Leuzzi, Rosanna; Kasendra, Magdalena; Minton, Nigel; Titball, Richard W; Michell, Stephen L

    2014-07-15

    Clostridium difficile is a cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease. Colonization of the gut is a critical step in the course of infection. The C. difficile lipoprotein CD0873 was identified as a putative adhesin through a bioinformatics approach. Surface exposure of CD0873 was confirmed and a CD0873 mutant was generated. The CD0873 mutant showed a significant reduction in adherence to Caco-2 cells and wild-type bacteria preincubated with anti-CD0873 antibodies showed significantly decreased adherence to Caco-2 cells. In addition, we demonstrated that purified recombinant CD0873 protein alone associates with Caco-2 cells. This is the first definitive identification of a C. difficile adhesin, which now allows work to devise improved measures for preventing and treating disease.

  14. Purifying protein complexes for mass spectrometry: applications to protein translation.

    PubMed

    Link, Andrew J; Fleischer, Tracey C; Weaver, Connie M; Gerbasi, Vincent R; Jennings, Jennifer L

    2005-03-01

    Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.

  15. The afimbrial adhesive sheath encoded by the afa-3 gene cluster of pathogenic Escherichia coli is composed of two adhesins.

    PubMed

    Garcia, M I; Gounon, P; Courcoux, P; Labigne, A; Le Bouguenec, C

    1996-02-01

    The afa-3 gene cluster determines the formation of an afimbrial adhesive sheath that is expressed by uropathogenic as well as diarrhoea-associated Escherichia coli strains. It contains six genes (afaA-afaF), among which the afaE3 gene is known to code for the structural AfaE-III adhesin (previously designated AFA-III), whereas no role has yet been identified for the afaD gene product. The afa-3 gene cluster is closely related to the daa operon that codes for an adhesin, the F1845 adhesin, which is highly related to the AfaE-III adhesin; however, unlike the AfaE-III adhesin, F1845 is a fimbrial adhesin. Reported in this work is the construction of chimeras between the afa-3 and daa operons. Analyses of the phenotypes conferred by these afa-3/daa chimeric clusters allowed us to conclude that the biogenesis of a fimbrial or an afimbrial adhesin is fully determined by the amino acid sequence of the AfaE-III and F1845 adhesins. Moreover, the role of the AfaD product in the biosynthesis of the afimbrial sheath was assessed by immunogold and immunofluorescence experiments. The AfaD and the AfaE-III products were purified and used to raise rabbit and mouse antisera. Similar to AfaE-III, AfaD was found to be a surface-exposed protein as well as an adhesin; both AfaD and AfaE-III are concomittantly expressed by the bacterial cell. These results demonstrate, for the first time, that the afimbrial adhesive sheath expressed by pathogenic E. coli is composed of two adhesins.

  16. The Human Disease-Associated Aβ Amyloid Core Sequence Forms Functional Amyloids in a Fungal Adhesin

    PubMed Central

    Rameau, Rachele D.; Jackson, Desmond N.; Beaussart, Audrey; Dufrêne, Yves F.

    2016-01-01

    ABSTRACT There is increasing evidence that many amyloids in living cells have physiological functions. On the surfaces of fungal cells, amyloid core sequences in adhesins can aggregate into 100- to 1,000-nm-wide patches to form high-avidity adhesion nanodomains on the cell surface. The nanodomains form through interactions that have amyloid-like properties: binding of amyloid dyes, perturbation by antiamyloid agents, and interaction with homologous sequences. To test whether these functional interactions are mediated by typical amyloid interactions, we substituted an amyloid core sequence, LVFFA, from human Aβ protein for the native sequence IVIVA in the 1,419-residue Candida albicans adhesin Als5p. The chimeric protein formed cell surface nanodomains and mediated cellular aggregation. The native sequence and chimeric adhesins responded similarly to the amyloid dye thioflavin T and to amyloid perturbants. However, unlike the native protein, the nanodomains formed by the chimeric protein were not force activated and formed less-robust aggregates under flow. These results showed the similarity of amyloid interactions in the amyloid core sequences of native Als5p and Aβ, but they also highlighted emergent properties of the native sequence. Also, a peptide composed of the Aβ amyloid sequence flanked by amino acids from the adhesin formed two-dimensional sheets with sizes similar to the cell surface patches of the adhesins. These results inform an initial model for the structure of fungal cell surface amyloid nanodomains. PMID:26758179

  17. Analysis of protein complexes using mass spectrometry.

    PubMed

    Gingras, Anne-Claude; Gstaiger, Matthias; Raught, Brian; Aebersold, Ruedi

    2007-08-01

    The versatile combination of affinity purification and mass spectrometry (AP-MS) has recently been applied to the detailed characterization of many protein complexes and large protein-interaction networks. The combination of AP-MS with other techniques, such as biochemical fractionation, intact mass measurement and chemical crosslinking, can help to decipher the supramolecular organization of protein complexes. AP-MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein-complex assembly.

  18. High resolution studies of the Afa/Dr adhesin DraE and its interaction with chloramphenicol.

    PubMed

    Pettigrew, David; Anderson, Kirstine L; Billington, Jason; Cota, Ernesto; Simpson, Peter; Urvil, Petri; Rabuzin, Filip; Roversi, Pietro; Nowicki, Bogdan; du Merle, Laurence; Le Bouguénec, Chantal; Matthews, Stephen; Lea, Susan M

    2004-11-05

    Pathogenic Escherichia coli expressing Afa/Dr adhesins are able to cause both urinary tract and diarrheal infections. The Afa/Dr adhesins confer adherence to epithelial cells via interactions with the human complement regulating protein, decay accelerating factor (DAF or CD55). Two of the Afa/Dr adhesions, AfaE-III and DraE, differ from each other by only three residues but are reported to have several different properties. One such difference is disruption of the interaction between DraE and CD55 by chloramphenicol, whereas binding of AfaE-III to CD55 is unaffected. Here we present a crystal structure of a strand-swapped trimer of wild type DraE. We also present a crystal structure of this trimer in complex with chloramphenicol, as well as NMR data supporting the binding position of chloramphenicol within the crystal. The crystal structure reveals the precise atomic basis for the sensitivity of DraE-CD55 binding to chloramphenicol and demonstrates that in contrast to other chloramphenicol-protein complexes, drug binding is mediated via recognition of the chlorine "tail" rather than via intercalation of the benzene rings into a hydrophobic pocket.

  19. Expression and purification of the mannose recognition domain of the FimH adhesin.

    PubMed

    Schembri, M A; Hasman, H; Klemm, P

    2000-07-15

    Type 1 fimbriae have been shown to be specifically required for Escherichia coli colonisation and pathogenesis of the urinary tract. These structural organelles mediate specific adhesion to alpha-D-mannosides by virtue of the FimH adhesin. FimH is a two-domain protein in which the N-terminal domain contains the receptor-binding site and the C-terminal domain is required for organelle integration. To date, FimH has only been isolated as a complex with the system-specific chaperone FimC. Here we report that a functional form of the FimH receptor-binding domain can be readily isolated and characterised by replacing the C-terminal domain with a histidine tag.

  20. Structural Studies of Protein-Surfactant Complexes

    SciTech Connect

    Chodankar, S. N.; Aswal, V. K.; Wagh, A. G.

    2008-03-17

    The structure of protein-surfactant complexes of two proteins bovine serum albumin (BSA) and lysozyme in presence of anionic surfactant sodium dodecyl sulfate (SDS) has been studied using small-angle neutron scattering (SANS). It is observed that these two proteins form different complex structures with the surfactant. While BSA protein undergoes unfolding on addition of surfactant, lysozyme does not show any unfolding even up to very high surfactant concentrations. The unfolding of BSA protein is caused by micelle-like aggregation of surfactant molecules in the complex. On the other hand, for lysozyme protein there is only binding of individual surfactant molecules to protein. Lysozyme in presence of higher surfactant concentrations has protein-surfactant complex structure coexisting with pure surfactant micelles.

  1. Trapping mammalian protein complexes in viral particles

    PubMed Central

    Eyckerman, Sven; Titeca, Kevin; Van Quickelberghe, Emmy; Cloots, Eva; Verhee, Annick; Samyn, Noortje; De Ceuninck, Leentje; Timmerman, Evy; De Sutter, Delphine; Lievens, Sam; Van Calenbergh, Serge; Gevaert, Kris; Tavernier, Jan

    2016-01-01

    Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. PMID:27122307

  2. A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction

    PubMed Central

    Chae, Keun; Gonong, Benedict J.; Kim, Seung-Chul; Kieslich, Chris A.; Morikis, Dimitrios; Balasubramanian, Shruthi; Lord, Elizabeth M.

    2010-01-01

    Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. β-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3 d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction. PMID:20667964

  3. Heterodimerization of the Entamoeba histolytica EhCPADH virulence complex through molecular dynamics and protein-protein docking.

    PubMed

    Montaño, Sarita; Orozco, Esther; Correa-Basurto, José; Bello, Martiniano; Chávez-Munguía, Bibiana; Betanzos, Abigail

    2017-02-01

    EhCPADH is a protein complex involved in the virulence of Entamoeba histolytica, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein-protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442-479 residues, adjacent to the target cell-adherence region (480-600 residues), and by the Bro1 domain (9-349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with in silico results.

  4. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  5. Co-translational assembly of protein complexes.

    PubMed

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2015-12-01

    The interaction of biological macromolecules is a fundamental attribute of cellular life. Proteins, in particular, often form stable complexes with one another. Although the importance of protein complexes is widely recognized, we still have only a very limited understanding of the mechanisms underlying their assembly within cells. In this article, we review the available evidence for one such mechanism, namely the coupling of protein complex assembly to translation at the polysome. We discuss research showing that co-translational assembly can occur in both prokaryotic and eukaryotic organisms and can have important implications for the correct functioning of the complexes that result. Co-translational assembly can occur for both homomeric and heteromeric protein complexes and for both proteins that are translated directly into the cytoplasm and those that are translated into or across membranes. Finally, we discuss the properties of proteins that are most likely to be associated with co-translational assembly.

  6. A Protein Complex Map of Trypanosoma brucei

    PubMed Central

    Mehta, Vaibhav; Najafabadi, Hamed S.; Moshiri, Houtan; Jardim, Armando; Salavati, Reza

    2016-01-01

    The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org. PMID:26991453

  7. Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

    PubMed

    Gómez-Molero, Emilia; de Boer, Albert D; Dekker, Henk L; Moreno-Martínez, Ana; Kraneveld, Eef A; Ichsan; Chauhan, Neeraj; Weig, Michael; de Soet, Johannes J; de Koster, Chris G; Bader, Oliver; de Groot, Piet W J

    2015-12-01

    Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.

  8. XadM, a novel adhesin of Xanthomonas oryzae pv. oryzae, exhibits similarity to Rhs family proteins and is required for optimum attachment, biofilm formation, and virulence.

    PubMed

    Pradhan, Binod B; Ranjan, Manish; Chatterjee, Subhadeep

    2012-09-01

    By screening a transposon-induced mutant library of Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, we have identified a novel 5.241-kb open reading frame (ORF) named xadM that is required for optimum virulence and colonization. This ORF encodes a protein, XadM, of 1,746 amino acids that exhibits significant similarity to Rhs family proteins. The XadM protein contains several repeat domains similar to a wall-associated surface protein of Bacillus subtilis, which has been proposed to be involved in carbohydrate binding. The role of XadM in X. oryzae pv. oryzae adhesion was demonstrated by the impaired ability of an xadM mutant strain to attach and form biofilms. Furthermore, we show that XadM is exposed on the cell surface and its expression is regulated by growth conditions and plays an important role in the early attachment and entry inside rice leaves. Interestingly, XadM homologs are present in several diverse bacteria, including many Xanthomonas spp. and animal-pathogenic bacteria belonging to Burkholderia spp. This is the first report of a role for XadM, an Rhs family protein, in adhesion and virulence of any pathogenic bacteria.

  9. A Protein Complex Network of Drosophila melanogaster

    PubMed Central

    Guruharsha, K. G.; Rual, J. -F.; Zhai, B.; Mintseris, J.; Vaidya, P.; Vaidya, N.; Beekman, C.; Wong, C.; Rhee, D. Y.; Cenaj, O.; McKillip, E.; Shah, S.; Stapleton, M.; Wan, K. H.; Yu, C.; Parsa, B.; Carlson, J. W.; Chen, X.; Kapadia, B.; VijayRaghavan, K.; Gygi, S. P.; Celniker, S. E.; Obar, R. A.; Artavanis-Tsakonas, S.

    2011-01-01

    SUMMARY Determining the composition of protein complexes is an essential step towards understanding the cell as an integrated system. Using co-affinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly five thousand individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a novel statistical framework to define individual protein-protein interactions, led to the generation of a Drosophila Protein interaction Map (DPiM) encompassing 556 protein complexes. The high quality of DPiM and its usefulness as a paradigm for metazoan proteomes is apparent from the recovery of many known complexes, significant enrichment for shared functional attributes and validation in human cells. DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution. PMID:22036573

  10. Neisseria Adhesin A Variation and Revised Nomenclature Scheme

    PubMed Central

    Bambini, Stefania; De Chiara, Matteo; Muzzi, Alessandro; Mora, Marirosa; Lucidarme, Jay; Brehony, Carina; Borrow, Ray; Masignani, Vega; Comanducci, Maurizio; Maiden, Martin C. J.; Pizza, Mariagrazia; Jolley, Keith A.

    2014-01-01

    Neisseria adhesin A (NadA), involved in the adhesion and invasion of Neisseria meningitidis into host tissues, is one of the major components of Bexsero, a novel multicomponent vaccine licensed for protection against meningococcal serogroup B in Europe, Australia, and Canada. NadA has been identified in approximately 30% of clinical isolates and in a much lower proportion of carrier isolates. Three protein variants were originally identified in invasive meningococci and named NadA-1, NadA-2, and NadA-3, whereas most carrier isolates either lacked the gene or harbored a different variant, NadA-4. Further analysis of isolates belonging to the sequence type 213 (ST-213) clonal complex identified NadA-5, which was structurally similar to NadA-4, but more distantly related to NadA-1, -2, and -3. At the time of this writing, more than 89 distinct nadA allele sequences and 43 distinct peptides have been described. Here, we present a revised nomenclature system, taking into account the complete data set, which is compatible with previous classification schemes and is expandable. The main features of this new scheme include (i) the grouping of the previously named NadA-2 and NadA-3 variants into a single NadA-2/3 variant, (ii) the grouping of the previously assigned NadA-4 and NadA-5 variants into a single NadA-4/5 variant, (iii) the introduction of an additional variant (NadA-6), and (iv) the classification of the variants into two main groups, named groups I and II. To facilitate querying of the sequences and submission of new allele sequences, the nucleotide and amino acid sequences are available at http://pubmlst.org/neisseria/NadA/. PMID:24807056

  11. Natural proteoglycan receptor analogs determine the dynamics of Opa adhesin-mediated gonococcal infection of Chang epithelial cells.

    PubMed Central

    van Putten, J P; Hayes, S F; Duensing, T D

    1997-01-01

    Many bacterial pathogens possess a complex machinery for the induction and/or secretion of factors that promote their uptake by mammalian cells. We searched for the molecular basis of the 60- to 90-min lag time in the interaction of Neisseria gonorrhoeae carrying the heparin-binding Opa adhesin with Chang epithelial cells. Infection assays in the presence of chloramphenicol demonstrated that the Opa-mediated gonococcal infection of Chang cells required bacterial protein synthesis when the microorganisms were derived from GC agar but not when grown in liquid media. Further analysis indicated that contact with agar ingredients rather than the growth state of the microorganisms determined the infection dynamics. DEAE chromatography of GC agar extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and testing of collected fractions in infection assays identified negatively charged high-molecular-weight polysaccharides in the agar as inhibitors of the cellular infection. Electron microscopy showed that agar-grown gonococci were surrounded by a coat of alcian blue-positive material, probably representing accreted polysaccharides. Similar antiphagocytic material was isolated from bovine serum, indicating that in biological fluids gonococci producing the heparin-binding Opa adhesin may become covered with externally derived polysaccharides as well. Binding assays with gonococci and epithelial proteoglycan receptors revealed that polysaccharides derived from agar or serum compete with the proteoglycans for binding of the heparin-binding Opa adhesin and thus act as receptor analogs. Growth of gonococci in a polysaccharide-free environment resulted in optimal proteoglycan receptor binding and rapid bacterial entry into Chang cells. The recognition that gonococci with certain phenotypes can recruit surface polysaccharides that determine in vitro infection dynamics adds a different dimension to the well-recognized biological significance of genetic

  12. [Isolation of proteins with complex forming agents].

    PubMed

    Schwenke, K D; Raab, B; Ender, B

    1975-01-01

    Taking vegetable albumins for models, the authors report of the possibilities of isolating proteins (which cannot be precipitated isoelectrically) by using their property of forming complexes with tannin or poly-anions. The precipitation of proteins with dextran sulphate or polyphosphates, which is due to electrostatic interaction, depends on the pH value and the electrolyte content of the solution. Under appropriate experimental conditions, protein yields of 100% are achieved. By means of tannin, the proteins are completely precipitated in a wide range of pH. The protein component of the poly-anion-containing complexes is isolated by precipitation with salt or by thermal coagulation after dissolving of the complexes. The isolation of protein from the tannin complexes is preferably realized by reaction with coffeine.

  13. FimH adhesin of Escherichia coli K1 type 1 fimbriae activates BV-2 microglia

    SciTech Connect

    Lee, Jongseok; Shin, Sooan; Teng, C.-H.; Hong, Suk Jin; Kim, Kwang Sik . E-mail: kwangkim@jhmi.edu

    2005-09-02

    The generation of intense inflammation in the subarachnoid space in response to meningitis-causing bacteria contributes to brain dysfunction and neuronal injury in bacterial meningitis. Microglia, the major immune effector cells in the central nervous system (CNS), become activated by bacterial components to produce proinflammatory immune mediators. In this study, we showed that FimH adhesin, a tip component of type 1 fimbriae of meningitis-causing Escherichia coli K1, activated the murine microglial cell line, BV-2, which resulted in the production of nitric oxide and the release of tumor necrosis factor-{alpha}. Mitogen-activated protein kinases, ERK and p-38, and nuclear factor-{kappa}B were involved in FimH adhesin-mediated microglial activation. These findings suggest that FimH adhesin contributes to the CNS inflammatory response by virtue of activating microglia in E. coli meningitis.

  14. Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Arnal, L.; Longo, G.; Stupar, P.; Castez, M. F.; Cattelan, N.; Salvarezza, R. C.; Yantorno, O. M.; Kasas, S.; Vela, M. E.

    2015-10-01

    Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract

  15. Sequence TTKF↓QE Defines the Site of Proteolytic Cleavage in Mhp683 Protein, a Novel Glycosaminoglycan and Cilium Adhesin of Mycoplasma hyopneumoniae*

    PubMed Central

    Bogema, Daniel R.; Scott, Nichollas E.; Padula, Matthew P.; Tacchi, Jessica L.; Raymond, Benjamin B. A.; Jenkins, Cheryl; Cordwell, Stuart J.; Minion, F. Chris; Walker, Mark J.; Djordjevic, Steven P.

    2011-01-01

    Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45683, P48683, and P50683. A peptide sequence (TTKF↓QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48683 and P50683, determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45683, P48683, and P50683 reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1683–F5683, spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1683–F5683 also bound porcine epithelial cilia, and antisera to F2683 and F5683 significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45683, P48683, and P50683 each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family. PMID:21969369

  16. Complex lasso: new entangled motifs in proteins

    NASA Astrophysics Data System (ADS)

    Niemyska, Wanda; Dabrowski-Tumanski, Pawel; Kadlof, Michal; Haglund, Ellinor; Sułkowski, Piotr; Sulkowska, Joanna I.

    2016-11-01

    We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules.

  17. Complex lasso: new entangled motifs in proteins

    PubMed Central

    Niemyska, Wanda; Dabrowski-Tumanski, Pawel; Kadlof, Michal; Haglund, Ellinor; Sułkowski, Piotr; Sulkowska, Joanna I.

    2016-01-01

    We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules. PMID:27874096

  18. Characterization of two new putative adhesins of Leptospira interrogans.

    PubMed

    Figueredo, Jupciana M; Siqueira, Gabriela H; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Chapola, Erica G B; Nascimento, Ana L T O

    2017-01-01

    We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

  19. Surfactant protein A (SP-A)-mediated clearance of Staphylococcus aureus involves binding of SP-A to the staphylococcal adhesin eap and the macrophage receptors SP-A receptor 210 and scavenger receptor class A.

    PubMed

    Sever-Chroneos, Zvjezdana; Krupa, Agnieszka; Davis, Jeremy; Hasan, Misbah; Yang, Ching-Hui; Szeliga, Jacek; Herrmann, Mathias; Hussain, Muzafar; Geisbrecht, Brian V; Kobzik, Lester; Chroneos, Zissis C

    2011-02-11

    Staphylococcus aureus causes life-threatening pneumonia in hospitals and deadly superinfection during viral influenza. The current study investigated the role of surfactant protein A (SP-A) in opsonization and clearance of S. aureus. Previous studies showed that SP-A mediates phagocytosis via the SP-A receptor 210 (SP-R210). Here, we show that SP-R210 mediates binding and control of SP-A-opsonized S. aureus by macrophages. We determined that SP-A binds S. aureus through the extracellular adhesin Eap. Consequently, SP-A enhanced macrophage uptake of Eap-expressing (Eap(+)) but not Eap-deficient (Eap(-)) S. aureus. In a reciprocal fashion, SP-A failed to enhance uptake of Eap(+) S. aureus in peritoneal Raw264.7 macrophages with a dominant negative mutation (SP-R210(DN)) blocking surface expression of SP-R210. Accordingly, WT mice cleared infection with Eap(+) but succumbed to sublethal infection with Eap- S. aureus. However, SP-R210(DN) cells compensated by increasing non-opsonic phagocytosis of Eap(+) S. aureus via the scavenger receptor scavenger receptor class A (SR-A), while non-opsonic uptake of Eap(-) S. aureus was impaired. Macrophages express two isoforms: SP-R210(L) and SP-R210(S). The results show that WT alveolar macrophages are distinguished by expression of SP-R210(L), whereas SR-A(-/-) alveolar macrophages are deficient in SP-R210(L) expressing only SP-R210(S). Accordingly, SR-A(-/-) mice were highly susceptible to both Eap(+) and Eap(-) S. aureus. The lungs of susceptible mice generated abnormal inflammatory responses that were associated with impaired killing and persistence of S. aureus infection in the lung. In conclusion, alveolar macrophage SP-R210(L) mediates recognition and killing of SP-A-opsonized S. aureus in vivo, coordinating inflammatory responses and resolution of S. aureus pneumonia through interaction with SR-A.

  20. Yersinia adhesins: An arsenal for infection.

    PubMed

    Chauhan, Nandini; Wrobel, Agnieszka; Skurnik, Mikael; Leo, Jack C

    2016-10-01

    The Yersiniae are a group of Gram-negative coccobacilli inhabiting a wide range of habitats. The genus harbors three recognized human pathogens: Y. enterocolitica and Y. pseudotuberculosis, which both cause gastrointestinal disease, and Y. pestis, the causative agent of plague. These three organisms have served as models for a number of aspects of infection biology, including adhesion, immune evasion, evolution of pathogenic traits, and retracing the course of ancient pandemics. The virulence of the pathogenic Yersiniae is heavily dependent on a number of adhesin molecules. Some of these, such as the Yersinia adhesin A and invasin of the enteropathogenic species, and the pH 6 antigen of Y. pestis, have been extensively studied. However, genomic sequencing has uncovered a host of other adhesins present in these organisms, the functions of which are only starting to be investigated. Here, we review the current state of knowledge on the adhesin molecules present in the Yersiniae, and their functions and putative roles in the infection process.

  1. Complementary Proteomic Analysis of Protein Complexes

    PubMed Central

    Greco, Todd M.; Miteva, Yana; Conlon, Frank L.; Cristea, Ileana M.

    2013-01-01

    Proteomic characterization of protein complexes leverages the versatile platform of liquid chromatography-tandem mass spectrometry to elucidate molecular and cellular signaling processes underlying the dynamic regulation of macromolecular assemblies. Here, we describe a complementary proteomic approach optimized for immunoisolated protein complexes. As the relative complexity, abundance, and physiochemical properties of proteins can vary significantly between samples, we have provided (1) complementary sample preparation workflows, (2) detailed steps for HPLC and mass spectrometric method development, and (3) a bioinformatic workflow that provides confident peptide/protein identification paired with unbiased functional gene ontology analysis. This protocol can also be extended for characterization of larger complexity samples from whole cell or tissue Xenopus proteomes. PMID:22956100

  2. The claudin Megatrachea protein complex.

    PubMed

    Jaspers, Martin H J; Nolde, Kai; Behr, Matthias; Joo, Seol-hee; Plessmann, Uwe; Nikolov, Miroslav; Urlaub, Henning; Schuh, Reinhard

    2012-10-26

    Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.

  3. Antibodies from multiple sclerosis patients preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae

    PubMed Central

    Walvoort, Marthe T. C.; Testa, Chiara; Eilam, Raya; Aharoni, Rina; Nuti, Francesca; Rossi, Giada; Real-Fernandez, Feliciana; Lanzillo, Roberta; Brescia Morra, Vincenzo; Lolli, Francesco; Rovero, Paolo; Imperiali, Barbara; Papini, Anna Maria

    2016-01-01

    In autoimmune diseases, there have been proposals that exogenous “molecular triggers”, i.e., specific this should be ‘non-self antigens’ accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of the multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS. PMID:28008952

  4. X-ray crystal structures of Staphylococcus aureus collagen adhesin and sortases

    NASA Astrophysics Data System (ADS)

    Zong, Yinong

    For many gram-positive bacteria, adhesion to host tissues is the first critical step in developing an infection. The adhesion is mediated by a superfamily of bacterial surface proteins, called MSCRAMM (microbial surface components recognizing adhesive matrix molecules), which in most cases are covalently attached to the cell wall peptidoglycan. Collagen adhesin (CNA) from Staphylococcus aureus, one of the MSCRAMMs, is responsible for bacterial binding to collagen molecules. CNA and other MSCRAMMs are anchored to the cell wall by a transpeptidase, sortase. The knowledge about how bacterial surface proteins adhere to host molecules and how they are sorted onto the cell wall is crucial for the design of novel antibiotics against bacterial infections. The crystal structures of CNA31--344 (residue 31 to 334), a truncation of CNA's collagen binding region, and CNA31--344 in complex with a collagen peptide were determined. CNA31--344 contains two domains, and between them is a big hole formed by a loop connecting the two domains. In the structure of CNA31--344-collagen complex, the collagen peptide is locked in the hole formed by the two domains of CNA 31--344. We reason that the two domains of CNA31--344 are open in the physiological condition, and close up when binding to collagen. This binding mechanism may be common for other bacterial collagen adhesins. There are two known sortases in Staphylococcus aureus. Sortase A is responsible for anchoring most MSCRAMMs that have a LPXTG (X represents any amino acid) sorting motif and sortase B for a bacterial ion acquisition protein. The crystal structures of both sortases indicate that they share a common catalytic mechanism. Unlike typical cysteine transpeptidases, sortases may use a novel Cys-Arg catalytic dyad instead of a Cys-His pair. All other sortases found in gram-positive bacteria may have similar active site architecture and employ the same catalytic dyad because the critical residues are all conserved among them

  5. Proteomics: bases for protein complexity understanding.

    PubMed

    Rotilio, Domenico; Della Corte, Anna; D'Imperio, Marco; Coletta, Walter; Marcone, Simone; Silvestri, Cristian; Giordano, Lucia; Di Michele, Michela; Donati, Maria Benedetta

    2012-03-01

    In the post genomic era we became aware that the genomic sequence and protein functions cannot be correlated. One gene can encode multiple protein functions mainly because of mRNA splice variants, post translational modifications (PTM) and moonlighting functions. To study the whole population of proteins present in a cell to a specific time point and under defined conditions it is necessary to investigate the proteome. Comprehensive analysis of the proteome requires the use of emerging high technologies because of the complexity and wide dynamic range of protein concentrations. Proteomics provides the tools to study protein identification and quantitation, protein-protein interactions, protein modifications and localization. The most widespread strategy for studying global protein expression employs two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) allowing thousands of proteins to be resolved and their expression quantified. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has emerged as a high throughput technique for protein identification and characterization because of its high sensitivity, precision and accuracy. LC-MS/MS is well suited for accurate quantitation of protein expression levels, post-translational modifications and comparative and absolute quantitative analysis of peptides. Bioinformatic tools are required to elaborate the growing number of proteomic data. Here, we give an overview of the current status of the wide range of technologies that define and characterize the modern proteomics.

  6. Heterologous Expression of Bartonella Adhesin A in Escherichia coli by Exchange of Trimeric Autotransporter Adhesin Domains Results in Enhanced Adhesion Properties and a Pathogenic Phenotype

    PubMed Central

    Schmidgen, Thomas; Kaiser, Patrick O.; Ballhorn, Wibke; Franz, Bettina; Göttig, Stephan; Linke, Dirk

    2014-01-01

    Human-pathogenic Bartonella henselae causes cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin (TAA) Bartonella adhesin A (BadA), which is modularly constructed, consisting of a head, a long and repetitive neck-stalk module, and a membrane anchor. BadA is involved in bacterial autoagglutination, binding to extracellular matrix proteins and host cells, and in proangiogenic reprogramming. The slow growth of B. henselae and limited tools for genetic manipulation are obstacles for detailed examination of BadA and its domains. Here, we established a recombinant expression system for BadA mutants in Escherichia coli allowing functional analysis of particular BadA domains. Using a BadA mutant lacking 21 neck-stalk repeats (BadA HN23), the BadA HN23 signal sequence was exchanged with that of E. coli OmpA, and the BadA membrane anchor was additionally replaced with that of Yersinia adhesin A (YadA). Constructs were cloned in E. coli, and hybrid protein expression was detected by immunoblotting, fluorescence microscopy, and flow cytometry. Functional analysis revealed that BadA hybrid proteins mediate autoagglutination and binding to collagen and endothelial cells. In vivo, expression of this BadA construct correlated with higher pathogenicity of E. coli in a Galleria mellonella infection model. PMID:24682330

  7. Protein-protein interactions in the synaptonemal complex.

    PubMed Central

    Tarsounas, M; Pearlman, R E; Gasser, P J; Park, M S; Moens, P B

    1997-01-01

    In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II. Images PMID:9285814

  8. Quantification of Detergents Complexed with Membrane Proteins

    PubMed Central

    Chaptal, Vincent; Delolme, Frédéric; Kilburg, Arnaud; Magnard, Sandrine; Montigny, Cédric; Picard, Martin; Prier, Charlène; Monticelli, Luca; Bornert, Olivier; Agez, Morgane; Ravaud, Stéphanie; Orelle, Cédric; Wagner, Renaud; Jawhari, Anass; Broutin, Isabelle; Pebay-Peyroula, Eva; Jault, Jean-Michel; Kaback, H. Ronald; le Maire, Marc; Falson, Pierre

    2017-01-01

    Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies. PMID:28176812

  9. Peroxisome protein import: a complex journey

    PubMed Central

    Baker, Alison; Hogg, Thomas Lanyon; Warriner, Stuart L.

    2016-01-01

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor–cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. PMID:27284042

  10. New Anthocyanin-Human Salivary Protein Complexes.

    PubMed

    Ferrer-Gallego, Raúl; Soares, Susana; Mateus, Nuno; Rivas-Gonzalo, Julián; Escribano-Bailón, M Teresa; de Freitas, Victor

    2015-08-04

    The interaction between phenolic compounds and salivary proteins is considered the basis of the poorly understood phenomenon of astringency. Furthermore, this interaction is an important factor in relation to their bioavailability. In this work, interactions between anthocyanin and human salivary protein fraction were studied by mass spectrometry (MALDI-TOF-MS and FIA-ESI-MS) and saturation-transfer difference (STD) NMR spectroscopy. Anthocyanins were able to interact with saliva proteins. The dissociation constant (KD) between malvidin 3-glucoside and salivary proline-rich proteins was 1.92 mM for the hemiketal form (pH 3.4) and 1.83 mM for the flavylium cation (pH 1.0). New soluble complexes between these salivary proteins and malvidin 3-glucoside were identified for the first time.

  11. Assembly reflects evolution of protein complexes.

    PubMed

    Levy, Emmanuel D; Boeri Erba, Elisabetta; Robinson, Carol V; Teichmann, Sarah A

    2008-06-26

    A homomer is formed by self-interacting copies of a protein unit. This is functionally important, as in allostery, and structurally crucial because mis-assembly of homomers is implicated in disease. Homomers are widespread, with 50-70% of proteins with a known quaternary state assembling into such structures. Despite their prevalence, their role in the evolution of cellular machinery and the potential for their use in the design of new molecular machines, little is known about the mechanisms that drive formation of homomers at the level of evolution and assembly in the cell. Here we present an analysis of over 5,000 unique atomic structures and show that the quaternary structure of homomers is conserved in over 70% of protein pairs sharing as little as 30% sequence identity. Where quaternary structure is not conserved among the members of a protein family, a detailed investigation revealed well-defined evolutionary pathways by which proteins transit between different quaternary structure types. Furthermore, we show by perturbing subunit interfaces within complexes and by mass spectrometry analysis, that the (dis)assembly pathway mimics the evolutionary pathway. These data represent a molecular analogy to Haeckel's evolutionary paradigm of embryonic development, where an intermediate in the assembly of a complex represents a form that appeared in its own evolutionary history. Our model of self-assembly allows reliable prediction of evolution and assembly of a complex solely from its crystal structure.

  12. Hsp70 Protein Complexes as Drug Targets

    PubMed Central

    Assimon, Victoria A.; Gillies, Anne T.; Rauch, Jennifer N.; Gestwicki, Jason E.

    2013-01-01

    Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70’s interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, such as pro-folding, pro-degradation and pro-trafficking. Thus, a promising strategy may be to block protein-protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to those goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology. PMID:22920901

  13. Hsp70 protein complexes as drug targets.

    PubMed

    Assimon, Victoria A; Gillies, Anne T; Rauch, Jennifer N; Gestwicki, Jason E

    2013-01-01

    Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70's interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, including roles in pro-folding, pro-degradation and pro-trafficking pathways. Thus, a promising strategy may be to block protein- protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to these goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology.

  14. AFA and F17 adhesins produced by pathogenic Escherichia coli strains in domestic animals.

    PubMed

    Le Bouguénec, C; Bertin, Y

    1999-01-01

    AFA and F17 are afimbrial and fimbrial adhesins, respectively, produced by pathogenic Escherichia coli strains in domestic animals. F17-related fimbriae are mainly detected on bovine and ovine E. coli associated with diarrhoea or septicaemia. The F17-G adhesin subunits recognize N-acetyl-D-glucosamine (GlcNAc) receptors present on bovine intestinal cells. Some F17 subtypes also bind to GlcNAc receptors present on human uroepithelial and intestinal Caco-2 cells or to the laminin contained in the basement of mammalian membranes. F17 is often associated with other virulence factors (aerobactin, serum resistance, CNF2 toxin, K99, CS31A or AFA adhesins) on pathogenic E. coli. A cluster of only four genes is required to synthesize functional F17-related fimbrial structures. The hypothesis of multifunctional F17 fimbrial subunits is supported by the fact that: i) the N-terminal part of the adhesin subunit participates in receptor recognition, whereas the C-terminal part is required for biogenesis of the fimbrial filament; and ii) the interaction between structural and adhesin subunits seems to be crucial for the initiation of monomer polymerization. Recently, determinants related to the afa gene clusters from human pathogenic E. coli associated with intestinal and extra-intestinal infections were identified in strains isolated from calves and piglets with diarrhoea and septicaemia. Two afa-related gene clusters, designated afa-7 and afa-8, that encode afimbrial adhesins were cloned and characterized from bovine pathogenic E. coli. These animal afa gene clusters were plasmid and chromosome borne and were expressed by strains that produced other virulence factors such as CNF toxins, F17, PAP and CS31A adhesins. A high frequency of afa-8 and a low prevalence of afa-7 among bovine E. coli isolates were suggested by preliminary epidemiological studies. As with the human afa gene clusters, the animal ones encode an adhesive structure composed of two proteins: AfaE which

  15. Characterization of protein complexes using targeted proteomics.

    PubMed

    Gomez, Yassel Ramos; Gallien, Sebastien; Huerta, Vivian; van Oostrum, Jan; Domon, Bruno; Gonzalez, Luis Javier

    2014-01-01

    Biological systems are not only controlled by the abundance of individual proteins, but also by the formation of complexes and the dynamics of protein-protein interactions. The identification of the components of protein complexes can be obtained by shotgun proteomics using affinity purification coupled to mass spectrometry. Such studies include the analyses of several samples and experimental controls in order to discriminate true specific interactions from unspecific interactions and contaminants. However, shotgun proteomics have limited quantification capabilities for low abundant proteins on large sample sets due to the undersampling and the stochastic precursor ion selection. In this context, targeted proteomics constitutes a powerful analytical tool to systematically detect and quantify peptides in multiple samples, for instance those obtained from affinity purification experiments. Hypothesis-driven strategies have mainly relied on the selected reaction monitoring (SRM) technique performed on triple quadrupole instruments, which enables highly selective and sensitive measurements of peptides, acting as surrogates of the pre-selected proteins, over a wide range of concentrations. More recently, novel quantitative methods based on high resolution instruments, such as the parallel reaction monitoring (PRM) technique implemented on the quadrupole-orbitrap instrument, have arisen and provided alternatives to perform quantitative analyses with enhanced selectivity.The application of targeted proteomics to protein-protein interaction experiments from plasma and other physiological fluid samples and the inclusion of parallel reaction monitoring (PRM), combined with other recent technology developments opens a vast area for clinical application of proteomics. It is anticipated that it will reveal valuable information about specific, individual, responses against drugs, exogenous proteins or pathogens.

  16. Interaction graph mining for protein complexes using local clique merging.

    PubMed

    Li, Xiao-Li; Tan, Soon-Heng; Foo, Chuan-Sheng; Ng, See-Kiong

    2005-01-01

    While recent technological advances have made available large datasets of experimentally-detected pairwise protein-protein interactions, there is still a lack of experimentally-determined protein complex data. To make up for this lack of protein complex data, we explore the mining of existing protein interaction graphs for protein complexes. This paper proposes a novel graph mining algorithm to detect the dense neighborhoods (highly connected regions) in an interaction graph which may correspond to protein complexes. Our algorithm first locates local cliques for each graph vertex (protein) and then merge the detected local cliques according to their affinity to form maximal dense regions. We present experimental results with yeast protein interaction data to demonstrate the effectiveness of our proposed method. Compared with other existing techniques, our predicted complexes can match or overlap significantly better with the known protein complexes in the MIPS benchmark database. Novel protein complexes were also predicted to help biologists in their search for new protein complexes.

  17. The soluble recombinant Neisseria meningitidis adhesin NadA(Δ351-405) stimulates human monocytes by binding to extracellular Hsp90.

    PubMed

    Cecchini, Paola; Tavano, Regina; Polverino de Laureto, Patrizia; Franzoso, Susanna; Mazzon, Cristina; Montanari, Paolo; Papini, Emanuele

    2011-01-01

    The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadA(Δ351-405,) devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadA(Δ351-405) cellular effects in monocytes. We show that NadA(Δ351-405) (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadA(Δ351-405) cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadA(Δ351-405) /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadA(Δ351-405) and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadA(Δ351-405) determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadA(Δ351-405) alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by

  18. Thermodynamics of interfacial changes in a protein-protein complex.

    PubMed

    Das, Amit; Chakrabarti, Jaydeb; Ghosh, Mahua

    2014-03-04

    Recent experiments with biomacromolecular complexes suggest that structural modifications at the interfaces are vital for stability of the complexes and the functions of the biomacromolecules. Although several qualitative aspects about such interfaces are known from structural data, quantification of the interfacial changes is lacking. In this work, we study the thermodynamic changes at the interface in the complex between an enzyme, Nuclease A (NucA), and a specific inhibitor protein, NuiA. We calculate the conformational free energy and conformational entropy costs from histograms of the dihedral angles generated from all-atom molecular dynamics simulations on the complex and the free proteins. We extract the conformational thermodynamic parameters for changes in the tertiary structure of NuiA. We show that the binding is dominated by the interfacial changes, where the basic residues of NucA and acidic residues of NuiA are highly ordered and stabilized via strong electrostatic interactions. Our results correlate well with known information from structural studies. The tight interfacial structure is reflected in the significant changes in the structure and dynamics of the water molecules at the enzyme-inhibitor interface. The interfacial water molecules contribute significantly to the entropy loss for the overall complexation.

  19. A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

    SciTech Connect

    Pyburn, Tasia M.; Bensing, Barbara A.; Xiong, Yan Q.; Melancon, Bruce J.; Tomasiak, Thomas M.; Ward, Nicholas J.; Yankovskaya, Victoria; Oliver, Kevin M.; Cecchini, Gary; Sulikowski, Gary A.; Tyska, Matthew J.; Sullam, Paul M.; Iverson, T.M.

    2014-10-02

    GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIb{alpha}. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB{sub BR}), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB{sub BR} structure revealed that it is comprised of three independently folded subdomains or modules: (1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; (2) a second Ig-fold resembling the binding region of mammalian Siglecs; (3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIb{alpha}. Further examination of purified GspB{sub BR}-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

  20. Oxygen promotes biofilm formation of Shewanella putrefaciens CN32 through a diguanylate cyclase and an adhesin

    PubMed Central

    Wu, Chao; Cheng, Yuan-Yuan; Yin, Hao; Song, Xiang-Ning; Li, Wen-Wei; Zhou, Xian-Xuan; Zhao, Li-Ping; Tian, Li-Jiao; Han, Jun-Cheng; Yu, Han-Qing

    2013-01-01

    Although oxygen has been reported to regulate biofilm formation by several Shewanella species, the exact regulatory mechanism mostly remains unclear. Here, we identify a direct oxygen-sensing diguanylate cyclase (DosD) and reveal its regulatory role in biofilm formation by Shewanella putrefaciens CN32 under aerobic conditions. In vitro and in vivo analyses revealed that the activity of DosD culminates to synthesis of cyclic diguanylate (c-di-GMP) in the presence of oxygen. DosD regulates the transcription of bpfA operon which encodes seven proteins including a large repetitive adhesin BpfA and its cognate type I secretion system (TISS). Regulation of DosD in aerobic biofilms is heavily dependent on an adhesin BpfA and the TISS. This study offers an insight into the molecular mechanism of oxygen-stimulated biofilm formation by S. putrefaciens CN32. PMID:23736081

  1. Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin.

    PubMed

    Bensing, Barbara A; Loukachevitch, Lioudmila V; McCulloch, Kathryn M; Yu, Hai; Vann, Kendra R; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M; Iverson, T M

    2016-04-01

    Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site.

  2. Architecture and adhesive activity of the Haemophilus influenzae Hsf adhesin.

    PubMed

    Cotter, Shane E; Yeo, Hye-Jeong; Juehne, Twyla; St Geme, Joseph W

    2005-07-01

    Haemophilus influenzae type b is an important cause of meningitis and other serious invasive diseases and initiates infection by colonizing the upper respiratory tract. Among the major adhesins in H. influenzae type b is a nonpilus protein called Hsf, a large protein that forms fiber-like structures on the bacterial surface and shares significant sequence similarity with the nontypeable H. influenzae Hia autotransporter. In the present study, we characterized the structure and adhesive activity of Hsf. Analysis of the predicted amino acid sequence of Hsf revealed three regions with high-level homology to the HiaBD1 and HiaBD2 binding domains in Hia. Based on examination of glutathione S-transferase fusion proteins corresponding to these regions, two of the three had adhesive activity and one was nonadhesive in assays with cultured epithelial cells. Structural modeling demonstrated that only the two regions with adhesive activity harbored an acidic binding pocket like the binding pocket identified in the crystal structure of HiaBD1. Consistent with these results, disruption of the acidic binding pockets in the adhesive regions eliminated adhesive activity. These studies advance our understanding of the architecture of Hsf and the family of trimeric autotransporters and provide insight into the structural determinants of H. influenzae type b adherence.

  3. Mapping the Binding Domain of the F18 Fimbrial Adhesin

    PubMed Central

    Smeds, A.; Pertovaara, M.; Timonen, T.; Pohjanvirta, T.; Pelkonen, S.; Palva, A.

    2003-01-01

    F18 fimbrial Esherichia coli strains are associated with porcine postweaning diarrhea and pig edema disease. Recently, the FedF subunit was identified as the adhesin of the F18 fimbriae. In this study, adhesion domains of FedF were further studied by constructing deletions within the fedF gene and expressing FedF proteins with deletions either together with the other F18 fimbrial subunits or as fusion proteins tagged with maltose binding protein. The region essential for adhesion to porcine intestinal epithelial cells was mapped between amino acid residues 60 and 109 of FedF. To map the binding domain even more closely, all eight charged amino acid residues within this region were independently replaced by alanine. Three of these single point mutants expressing F18 fimbriae exhibited significantly diminished capabilities to adhere to porcine epithelial cells in vitro. In addition, a triple point mutation and a double point mutation completely abolished receptor adhesiveness. The result further confirmed that the region between amino acid residues 60 and 109 is essential for the binding of F18 fimbriae to their receptor. In addition, the adhesion capability of the binding domain was eliminated after treatment with iodoacetamide, suggesting the formation of a disulfide bridge between Cys-63 and Cys-83, whereas Cys-111 and Cys-116 could be deleted without affecting the binding ability of FedF. PMID:12654838

  4. Analysis of DNA-protein complexes induced by chemical carcinogens

    SciTech Connect

    Costa, M. )

    1990-11-01

    DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.

  5. Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

    PubMed

    Silva, Carlos A M; Danelishvili, Lia; McNamara, Michael; Berredo-Pinho, Márcia; Bildfell, Robert; Biet, Franck; Rodrigues, Luciana S; Oliveira, Albanita V; Bermudez, Luiz E; Pessolani, Maria C V

    2013-07-01

    This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

  6. Haemophilus influenzae protein E recognizes the C-terminal domain of vitronectin and modulates the membrane attack complex.

    PubMed

    Singh, Birendra; Jalalvand, Farshid; Mörgelin, Matthias; Zipfel, Peter; Blom, Anna M; Riesbeck, Kristian

    2011-07-01

    Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune response in lung epithelial cells. The active epithelial binding region comprising amino acids PE 84-108 also interferes with complement-mediated bacterial killing by capturing vitronectin (Vn) that prevents complement deposition and formation of the membrane attack complex (MAC). Here, the interaction between PE and Vn was characterized using site-directed mutagenesis. Protein E variants were produced both in soluble forms and in surface-expressed molecules on Escherichia coli. Mutations within PE(84-108) in the full-length molecule revealed that K85 and R86 residues were important for the Vn binding. Bactericidal activity against H. influenzae was higher in human serum pre-treated with full-length PE as compared with serum incubated with PE(K85E, R86D) , suggesting that PE quenched Vn. A series of truncated Vn molecules revealed that the C-terminal domain comprising Vn(353-363) harboured the major binding region for PE. Interestingly, MAC deposition was significantly higher on mutants devoid of PE due to a decreased Vn-binding capacity when compared with wild-type H. influenzae. Our results define a fine-tuned interaction between H. influenzae and the innate immune system, and identify the mode of control of the MAC that is important for pathogen complement evasion.

  7. Engineering of complex protein sialylation in plants

    PubMed Central

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-01-01

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  8. An Acinetobacter trimeric autotransporter adhesin reaped from cells exhibits its nonspecific stickiness via a highly stable 3D structure

    PubMed Central

    Yoshimoto, Shogo; Nakatani, Hajime; Iwasaki, Keita; Hori, Katsutoshi

    2016-01-01

    Trimeric autotransporter adhesins (TAAs), cell surface proteins of Gram-negative bacteria, mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific, high adhesiveness to abiotic material surfaces as well as to biotic surfaces. AtaA is a homotrimer of polypeptides comprising 3,630 amino acids and forms long nanofibers; therefore, it is too large and structurally complex to be produced as a recombinant protein. In this study, we isolated AtaA’s passenger domain (AtaA PSD), which is translocated to the cell surface through the C-terminal transmembrane domain and exhibits biological functions, using a new method. We introduced a protease recognition site and reaped AtaA nanofibers 225 nm in length from the cell surface through proteolytic cleavage with a specific protease. Biochemical and biophysical analyses of the purified native AtaA PSD revealed that it has a stable structure under alkaline and acidic conditions. Temperatures above 80 °C, which disrupted AtaA’s higher-order structure but maintained the full-length AtaA polypeptide, inactivated AtaA’s nonspecific adhesiveness, suggesting that the stickiness of AtaA requires its 3D structure. This finding refutes the widespread but vague speculation that large unfolded polypeptides readily stick to various surfaces. PMID:27305955

  9. Detecting overlapping protein complexes by rough-fuzzy clustering in protein-protein interaction networks.

    PubMed

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks.

  10. The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A.

    PubMed

    Ragas, Aude; Roussel, Lucie; Puzo, Germain; Rivière, Michel

    2007-02-23

    Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional

  11. T4 Phage Tail Adhesin Gp12 Counteracts LPS-Induced Inflammation In Vivo

    PubMed Central

    Miernikiewicz, Paulina; Kłopot, Anna; Soluch, Ryszard; Szkuta, Piotr; Kęska, Weronika; Hodyra-Stefaniak, Katarzyna; Konopka, Agnieszka; Nowak, Marcin; Lecion, Dorota; Kaźmierczak, Zuzanna; Majewska, Joanna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2016-01-01

    Bacteriophages that infect Gram-negative bacteria often bind to the bacterial surface by interaction of specific proteins with lipopolysaccharide (LPS). Short tail fiber proteins (tail adhesin, gp12) mediate adsorption of T4-like bacteriophages to Escherichia coli, binding surface proteins or LPS. Produced as a recombinant protein, gp12 retains its ability to bind LPS. Since LPS is able to exert a major impact on the immune response in animals and in humans, we have tested LPS-binding phage protein gp12 as a potential modulator of the LPS-induced immune response. We have produced tail adhesin gp12 in a bacterial expression system and confirmed its ability to form trimers and to bind LPS in vitro by dynamic light scattering. This product had no negative effect on mammalian cell proliferation in vitro. Further, no harmful effects of this protein were observed in mice. Thus, gp12 was used in combination with LPS in a murine model, and it decreased the inflammatory response to LPS in vivo, as assessed by serum levels of cytokines IL-1 alpha and IL-6 and by histopathological analysis of spleen, liver, kidney and lungs. Thus, in future studies gp12 may be considered as a potential tool for modulating and specifically for counteracting LPS-related physiological effects in vivo. PMID:27471503

  12. Construction of ontology augmented networks for protein complex prediction.

    PubMed

    Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian

    2013-01-01

    Protein complexes are of great importance in understanding the principles of cellular organization and function. The increase in available protein-protein interaction data, gene ontology and other resources make it possible to develop computational methods for protein complex prediction. Most existing methods focus mainly on the topological structure of protein-protein interaction networks, and largely ignore the gene ontology annotation information. In this article, we constructed ontology augmented networks with protein-protein interaction data and gene ontology, which effectively unified the topological structure of protein-protein interaction networks and the similarity of gene ontology annotations into unified distance measures. After constructing ontology augmented networks, a novel method (clustering based on ontology augmented networks) was proposed to predict protein complexes, which was capable of taking into account the topological structure of the protein-protein interaction network, as well as the similarity of gene ontology annotations. Our method was applied to two different yeast protein-protein interaction datasets and predicted many well-known complexes. The experimental results showed that (i) ontology augmented networks and the unified distance measure can effectively combine the structure closeness and gene ontology annotation similarity; (ii) our method is valuable in predicting protein complexes and has higher F1 and accuracy compared to other competing methods.

  13. Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

    PubMed Central

    Edwards, Thomas E.; Phan, Isabelle; Abendroth, Jan; Dieterich, Shellie H.; Masoudi, Amir; Guo, Wenjin; Hewitt, Stephen N.; Kelley, Angela; Leibly, David; Brittnacher, Mitch J.; Staker, Bart L.; Miller, Samuel I.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.

    2010-01-01

    Background Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region. Methodology/Principal Findings Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 Å resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs. Conclusions/Significance The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics. PMID:20862217

  14. 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

  15. Separation of membrane protein complexes by native LDS-PAGE.

    PubMed

    Arnold, Janine; Shapiguzov, Alexey; Fucile, Geoffrey; Rochaix, Jean-David; Goldschmidt-Clermont, Michel; Eichacker, Lutz Andreas

    2014-01-01

    Gel electrophoresis has become one of the most important methods for the analysis of proteins and protein complexes in a molecular weight range of 1-10(7) kDa. The separation of membrane protein complexes remained challenging to standardize until the demonstration of Blue Native PAGE in 1991 [1] and Clear Native PAGE in 1994 [2]. We present a robust protocol for high-resolution separation of photosynthetic complexes from Arabidopsis thaliana using lithium dodecyl sulfate as anion in a modified Blue Native PAGE (LDS-PAGE). Here, non-covalently bound chlorophyll is used as a sensitive probe to characterize the assembly/biogenesis of the pigment-protein complexes essential for photosynthesis. The high fluorescence yield recorded from chlorophyll-binding protein complexes can also be used to establish the separation of native protein complexes as an electrophoretic standard.

  16. Structure, dynamics, assembly, and evolution of protein complexes.

    PubMed

    Marsh, Joseph A; Teichmann, Sarah A

    2015-01-01

    The assembly of individual proteins into functional complexes is fundamental to nearly all biological processes. In recent decades, many thousands of homomeric and heteromeric protein complex structures have been determined, greatly improving our understanding of the fundamental principles that control symmetric and asymmetric quaternary structure organization. Furthermore, our conception of protein complexes has moved beyond static representations to include dynamic aspects of quaternary structure, including conformational changes upon binding, multistep ordered assembly pathways, and structural fluctuations occurring within fully assembled complexes. Finally, major advances have been made in our understanding of protein complex evolution, both in reconstructing evolutionary histories of specific complexes and in elucidating general mechanisms that explain how quaternary structure tends to evolve. The evolution of quaternary structure occurs via changes in self-assembly state or through the gain or loss of protein subunits, and these processes can be driven by both adaptive and nonadaptive influences.

  17. Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects

    PubMed Central

    Arciola, Carla Renata; Campoccia, Davide; Ravaioli, Stefano; Montanaro, Lucio

    2015-01-01

    Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to ica locus are present in many coagulase-negative staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm production have been described. S. epidermidis and S. aureus undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection. PMID

  18. Hysteresis as a Marker for Complex, Overlapping Landscapes in Proteins

    PubMed Central

    Andrews, Benjamin T.; Capraro, Dominique T.; Sulkowska, Joanna I.; Onuchic, José N.; Jennings, Patricia A.

    2013-01-01

    Topologically complex proteins fold by multiple routes as a result of hard-to-fold regions of the proteins. Oftentimes these regions are introduced into the protein scaffold for function and increase frustration in the otherwise smooth-funneled landscape. Interestingly, while functional regions add complexity to folding landscapes, they may also contribute to a unique behavior referred to as hysteresis. While hysteresis is predicted to be rare, it is observed in various proteins, including proteins containing a unique peptide cyclization to form a fluorescent chromophore as well as proteins containing a knotted topology in their native fold. Here, hysteresis is demonstrated to be a consequence of the decoupling of unfolding events from the isomerization or hula-twist of a chromophore in one protein and the untying of the knot in a second protein system. The question now is- can hysteresis be a marker for the interplay of landscapes where complex folding and functional regions overlap? PMID:23525263

  19. Biodiversity-Based Identification and Functional Characterization of the Mannose-Specific Adhesin of Lactobacillus plantarum

    PubMed Central

    Pretzer, Gabriele; Snel, Johannes; Molenaar, Douwe; Wiersma, Anne; Bron, Peter A.; Lambert, Jolanda; de Vos, Willem M.; van der Meer, Roelof; Smits, Mari A.; Kleerebezem, Michiel

    2005-01-01

    Lactobacillus plantarum is a frequently encountered inhabitant of the human intestinal tract, and some strains are marketed as probiotics. Their ability to adhere to mannose residues is a potentially interesting characteristic with regard to proposed probiotic features such as colonization of the intestinal surface and competitive exclusion of pathogens. In this study, the variable capacity of 14 L. plantarum strains to agglutinate Saccharomyces cerevisiae in a mannose-specific manner was determined and subsequently correlated with an L. plantarum WCFS1-based genome-wide genotype database. This led to the identification of four candidate mannose adhesin-encoding genes. Two genes primarily predicted to code for sortase-dependent cell surface proteins displayed a complete gene-trait match. Their involvement in mannose adhesion was corroborated by the finding that a sortase (srtA) mutant of L. plantarum WCFS1 lost the capacity to agglutinate S. cerevisiae. The postulated role of these two candidate genes was investigated by gene-specific deletion and overexpression in L. plantarum WCFS1. Subsequent evaluation of the mannose adhesion capacity of the resulting mutant strains showed that inactivation of one candidate gene (lp_0373) did not affect mannose adhesion properties. In contrast, deletion of the other gene (lp_1229) resulted in a complete loss of yeast agglutination ability, while its overexpression quantitatively enhanced this phenotype. Therefore, this gene was designated to encode the mannose-specific adhesin (Msa; gene name, msa) of L. plantarum. Domain homology analysis of the predicted 1,000-residue Msa protein identified known carbohydrate-binding domains, further supporting its role as a mannose adhesin that is likely to be involved in the interaction of L. plantarum with its host in the intestinal tract. PMID:16109954

  20. A proteomic strategy for global analysis of plant protein complexes.

    PubMed

    Aryal, Uma K; Xiong, Yi; McBride, Zachary; Kihara, Daisuke; Xie, Jun; Hall, Mark C; Szymanski, Daniel B

    2014-10-01

    Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.

  1. Design and characterization of complex protein films

    NASA Astrophysics Data System (ADS)

    Bui, Holt P.

    Once a biomaterial is implanted into biological system, a layer of protein is immediately deposited on the surface of that material. The newly formed protein film will dictate how the implanted material will interact with the surrounding biological environment and lead to either the acceptance or rejection of the biomaterial. One method to enhance performance involves the activation the surface of the biomaterial with one or more proteins to direct specific interactions with the host environment. The focus of my dissertation was to develop and characterize model biomaterials surfaces that are activated with one or more proteins to help understand how the protein films may affect biological processes and a biomaterial's performance. One model system consisted of a patterned film of two proteins on a gold surface. Characterization of this protein pattern indicated that patterning protein films with a focused ion beam produced protein patterns with high biological contrast and high spatial control. The second model protein film involved the adsorption of fibronectin on surfaces with different surface energies. The characterization of the adsorbed fibronectin films suggest that fibronectin adsorbed on a hydrophilic surface is in an orientation that projects hydrophilic amino acid residues towards surface of the protein and dehydration causes reorientation to project hydrophobic amino acids towards the surface. In contrast, fibronectin is adsorbed onto a hydrophobic surface in a manner that resulted in dehydration and denaturation during the adsorption process. The last model protein film studied in this work consisted of fibronectin patterned in a manner so that the film consisted of spatially controlled domains of fibronectin adsorbed onto a hydrophilic surface as well as a hydrophobic surface. Lateral characterization of this pattern demonstrated a difference in secondary structure of fibronectin adsorbed on the two domains with varying surface energies.

  2. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

    PubMed

    Krogan, Nevan J; Cagney, Gerard; Yu, Haiyuan; Zhong, Gouqing; Guo, Xinghua; Ignatchenko, Alexandr; Li, Joyce; Pu, Shuye; Datta, Nira; Tikuisis, Aaron P; Punna, Thanuja; Peregrín-Alvarez, José M; Shales, Michael; Zhang, Xin; Davey, Michael; Robinson, Mark D; Paccanaro, Alberto; Bray, James E; Sheung, Anthony; Beattie, Bryan; Richards, Dawn P; Canadien, Veronica; Lalev, Atanas; Mena, Frank; Wong, Peter; Starostine, Andrei; Canete, Myra M; Vlasblom, James; Wu, Samuel; Orsi, Chris; Collins, Sean R; Chandran, Shamanta; Haw, Robin; Rilstone, Jennifer J; Gandi, Kiran; Thompson, Natalie J; Musso, Gabe; St Onge, Peter; Ghanny, Shaun; Lam, Mandy H Y; Butland, Gareth; Altaf-Ul, Amin M; Kanaya, Shigehiko; Shilatifard, Ali; O'Shea, Erin; Weissman, Jonathan S; Ingles, C James; Hughes, Timothy R; Parkinson, John; Gerstein, Mark; Wodak, Shoshana J; Emili, Andrew; Greenblatt, Jack F

    2006-03-30

    Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

  3. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering.

  4. Recording information on protein complexes in an information management system.

    PubMed

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described.

  5. Multi-LZerD: Multiple protein docking for asymmetric complexes

    PubMed Central

    Esquivel-Rodríguez, Juan; Yang, Yifeng David; Kihara, Daisuke

    2012-01-01

    The tertiary structures of protein complexes provide a crucial insight about the molecular mechanisms that regulate their functions and assembly. However, solving protein complex structures by experimental methods is often more difficult than single protein structures. Here, we have developed a novel computational multiple protein docking algorithm, Multi-LZerD, that builds models of multimeric complexes by effectively reusing pairwise docking predictions of component proteins. A genetic algorithm is applied to explore the conformational space followed by a structure refinement procedure. Benchmark on eleven hetero-multimeric complexes resulted in near native conformations for all but one of them (a root mean square deviation smaller than 2.5Å). We also show that our method copes with unbound docking cases well, outperforming the methodology that can be directly compared to our approach. Multi-LZerD was able to predict near native structures for multimeric complexes of various topologies. PMID:22488467

  6. Native Mass Spectrometry of Photosynthetic Pigment-Protein Complexes

    PubMed Central

    Zhang, Hao; Cui, Weidong; Gross, Michael L.; Blankenship, Robert E.

    2013-01-01

    Native mass spectrometry, or as is sometimes called “native electrospray (ESI)” allows proteins in their native or near-native protein in solution to be introduced into gas phase and interrogated by MS. This approach is now a powerful tool to investigate protein complexes. This article reviews the background of native MS of protein complexes and describes its strengths, taking photosynthetic pigment-protein complexes as examples. Native MS can be utilized in combination with other MS-based approaches to obtain complementary information to that provided by tools such as X-ray crystallography and NMR spectroscopy to understand the structure-function relationships of protein complexes. When additional information beyond that provided by native MS is required, other MS-based strategies can be successfully applied to augment the results of native MS. PMID:23337874

  7. Advances in protein complex analysis using mass spectrometry

    PubMed Central

    Gingras, Anne-Claude; Aebersold, Ruedi; Raught, Brian

    2005-01-01

    Proteins often function as components of larger complexes to perform a specific function, and formation of these complexes may be regulated. For example, intracellular signalling events often require transient and/or regulated protein–protein interactions for propagation, and protein binding to a specific DNA sequence, RNA molecule or metabolite is often regulated to modulate a particular cellular function. Thus, characterizing protein complexes can offer important insights into protein function. This review describes recent important advances in mass spectrometry (MS)-based techniques for the analysis of protein complexes. Following brief descriptions of how proteins are identified using MS, and general protein complex purification approaches, we address two of the most important issues in these types of studies: specificity and background protein contaminants. Two basic strategies for increasing specificity and decreasing background are presented: whereas (1) tandem affinity purification (TAP) of tagged proteins of interest can dramatically improve the signal-to-noise ratio via the generation of cleaner samples, (2) stable isotopic labelling of proteins may be used to discriminate between contaminants and bona fide binding partners using quantitative MS techniques. Examples, as well as advantages and disadvantages of each approach, are presented. PMID:15611014

  8. UpaG, a New Member of the Trimeric Autotransporter Family of Adhesins in Uropathogenic Escherichia coli▿ †

    PubMed Central

    Valle, Jaione; Mabbett, Amanda N.; Ulett, Glen C.; Toledo-Arana, Alejandro; Wecker, Karine; Totsika, Makrina; Schembri, Mark A.; Ghigo, Jean-Marc; Beloin, Christophe

    2008-01-01

    The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins. PMID:18424525

  9. Luminogenic "clickable" lanthanide complexes for protein labeling.

    PubMed

    Candelon, Nicolas; Hădade, Niculina D; Matache, Mihaela; Canet, Jean-Louis; Cisnetti, Federico; Funeriu, Daniel P; Nauton, Lionel; Gautier, Arnaud

    2013-10-14

    Development of lanthanide-based luminescent "switch-on" systems via azide-alkyne [3+2] cycloaddition is described. We used these for non-specific protein labeling and as tags for specific and selective activity-based protein labeling.

  10. Multiscale Model for the Assembly Kinetics of Protein Complexes.

    PubMed

    Xie, Zhong-Ru; Chen, Jiawen; Wu, Yinghao

    2016-02-04

    The assembly of proteins into high-order complexes is a general mechanism for these biomolecules to implement their versatile functions in cells. Natural evolution has developed various assembling pathways for specific protein complexes to maintain their stability and proper activities. Previous studies have provided numerous examples of the misassembly of protein complexes leading to severe biological consequences. Although the research focusing on protein complexes has started to move beyond the static representation of quaternary structures to the dynamic aspect of their assembly, the current understanding of the assembly mechanism of protein complexes is still largely limited. To tackle this problem, we developed a new multiscale modeling framework. This framework combines a lower-resolution rigid-body-based simulation with a higher-resolution Cα-based simulation method so that protein complexes can be assembled with both structural details and computational efficiency. We applied this model to a homotrimer and a heterotetramer as simple test systems. Consistent with experimental observations, our simulations indicated very different kinetics between protein oligomerization and dimerization. The formation of protein oligomers is a multistep process that is much slower than dimerization but thermodynamically more stable. Moreover, we showed that even the same protein quaternary structure can have very diverse assembly pathways under different binding constants between subunits, which is important for regulating the functions of protein complexes. Finally, we revealed that the binding between subunits in a complex can be synergistically strengthened during assembly without considering allosteric regulation or conformational changes. Therefore, our model provides a useful tool to understand the general principles of protein complex assembly.

  11. Protein-protein interactions in complex cosolvent solutions.

    PubMed

    Javid, Nadeem; Vogtt, Karsten; Krywka, Chris; Tolan, Metin; Winter, Roland

    2007-04-02

    The effects of various kosmotropic and chaotropic cosolvents and salts on the intermolecular interaction potential of positively charged lysozyme is evaluated at varying protein concentrations by using synchrotron small-angle X-ray scattering in combination with liquid-state theoretical approaches. The experimentally derived static structure factors S(Q) obtained without and with added cosolvents and salts are analysed with a statistical mechanical model based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) potential, which accounts for repulsive and attractive interactions between the protein molecules. Different cosolvents and salts influence the interactions between protein molecules differently as a result of changes in the hydration level or solvation, in charge screening, specific adsorption of the additives at the protein surface, or increased hydrophobic interactions. Intermolecular interaction effects are significant above protein concentrations of 1 wt %, and with increasing protein concentration, the repulsive nature of the intermolecular pair potential V(r) increases markedly. Kosmotropic cosolvents like glycerol and sucrose exhibit strong concentration-dependent effects on the interaction potential, leading to an increase of repulsive forces between the protein molecules at low to medium high osmolyte concentrations. Addition of trifluoroethanol exhibits a multiphasic effect on V(r) when changing its concentration. Salts like sodium chloride and potassium sulfate exhibit strong concentration-dependent changes of the interaction potential due to charge screening of the positively charged protein molecules. Guanidinium chloride (GdmCl) at low concentrations exhibits a similar charge-screening effect, resulting in increased attractive interactions between the protein molecules. At higher GdmCl concentrations, V(r) becomes more repulsive in nature due to the presence of high concentrations of Gdm(+) ions binding to the protein molecules. Our findings also

  12. Identifying protein complexes in protein-protein interaction networks by using clique seeds and graph entropy.

    PubMed

    Chen, Bolin; Shi, Jinhong; Zhang, Shenggui; Wu, Fang-Xiang

    2013-01-01

    The identification of protein complexes plays a key role in understanding major cellular processes and biological functions. Various computational algorithms have been proposed to identify protein complexes from protein-protein interaction (PPI) networks. In this paper, we first introduce a new seed-selection strategy for seed-growth style algorithms. Cliques rather than individual vertices are employed as initial seeds. After that, a result-modification approach is proposed based on this seed-selection strategy. Predictions generated by higher order clique seeds are employed to modify results that are generated by lower order ones. The performance of this seed-selection strategy and the result-modification approach are tested by using the entropy-based algorithm, which is currently the best seed-growth style algorithm to detect protein complexes from PPI networks. In addition, we investigate four pairs of strategies for this algorithm in order to improve its accuracy. The numerical experiments are conducted on a Saccharomyces cerevisiae PPI network. The group of best predictions consists of 1711 clusters, with the average f-score at 0.68 after removing all similar and redundant clusters. We conclude that higher order clique seeds can generate predictions with higher accuracy and that our improved entropy-based algorithm outputs more reasonable predictions than the original one.

  13. Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii.

    PubMed

    Lima, Bruno P; Shi, Wenyuan; Lux, Renate

    2017-02-07

    To successfully colonize the oral cavity, bacteria must directly or indirectly adhere to available oral surfaces. Fusobacterium nucleatum plays an important role in oral biofilm community development due to its broad adherence abilities, serving as a bridge between members of the oral biofilm that cannot directly bind to each other. In our efforts to characterize the molecular mechanisms utilized by F. nucleatum to physically bind to key members of the oral community, we investigated the involvement of F. nucleatum outer membrane proteins in its ability to bind to the pioneer biofilm colonizer, Streptococcus gordonii. Here, we present evidence that in addition to the previously characterized fusobacterial adhesin RadD, the interaction between F. nucleatum ATCC 23726 and S. gordonii V288 involves a second outer membrane protein, which we named coaggregation mediating protein A (CmpA). We also characterized the role of CmpA in dual-species biofilm formation with S. gordonii V288, evaluated growth-phase-dependent as well as biofilm expression profiles of radD and cmpA, and confirmed an important role for CmpA, especially under biofilm growth conditions. Our findings underscore the complex set of specific interactions involved in physical binding and thus community integration of interacting bacterial species. This complex set of interactions could have critical implications for the formation and maturation of the oral biofilms in vivo, and could provide clues to the mechanism behind the distribution of organisms inside the human oral cavity.

  14. Measurement of protein-ligand complex formation.

    PubMed

    Lowe, Peter N; Vaughan, Cara K; Daviter, Tina

    2013-01-01

    Experimental approaches to detect, measure, and quantify protein-ligand binding, along with their theoretical bases, are described. A range of methods for detection of protein-ligand interactions is summarized. Specific protocols are provided for a nonequilibrium procedure pull-down assay, for an equilibrium direct binding method and its modification into a competition-based measurement and for steady-state measurements based on the effects of ligands on enzyme catalysis.

  15. Recording information on protein complexes in an information management system

    PubMed Central

    Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

    2011-01-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  16. Conservation of Telomere protein complexes: Shuffling through Evolution

    PubMed Central

    Linger, Benjamin R.; Price, Carolyn M.

    2009-01-01

    The rapid evolution of telomere proteins has hindered identification of orthologs from diverse species and created the impression that certain groups of eukaryotes have largely non-overlapping sets of telomere proteins. However, the recent identification of additional telomere proteins from various model organisms has dispelled this notion by expanding our understanding of the composition, architecture and range of telomere protein complexes present in individual species. It is now apparent that versions of the budding yeast CST complex and mammalian shelterin are present in multiple phyla. While the precise subunit composition and architecture of these complexes vary between species, the general function is often conserved. Despite the overall conservation of telomere protein complexes, there is still considerable species specific variation, with some organisms having lost a particular subunit or even an entire complex. In some cases, complex components appear to have migrated between the telomere and the telomerase RNP. Finally, gene duplication has created telomere protein paralogs with novel functions. While one paralog may be part of a conserved telomere protein complex and have the expected function, the other paralog may serve in a completely different aspect of telomere biology. PMID:19839711

  17. The retromer complex - endosomal protein recycling and beyond.

    PubMed

    Seaman, Matthew N J

    2012-10-15

    The retromer complex is a vital element of the endosomal protein sorting machinery that is conserved across all eukaryotes. Retromer is most closely associated with the endosome-to-Golgi retrieval pathway and is necessary to maintain an active pool of hydrolase receptors in the trans-Golgi network. Recent progress in studies of retromer have identified new retromer-interacting proteins, including the WASH complex and cargo such as the Wntless/MIG-14 protein, which now extends the role of retromer beyond the endosome-to-Golgi pathway and has revealed that retromer is required for aspects of endosome-to-plasma membrane sorting and regulation of signalling events. The interactions between the retromer complex and other macromolecular protein complexes now show how endosomal protein sorting is coordinated with actin assembly and movement along microtubules, and place retromer squarely at the centre of a complex set of protein machinery that governs endosomal protein sorting. Dysregulation of retromer-mediated endosomal protein sorting leads to various pathologies, including neurodegenerative diseases such as Alzheimer disease and spastic paraplegia and the mechanisms underlying these pathologies are starting to be understood. In this Commentary, I will highlight recent advances in the understanding of retromer-mediated endosomal protein sorting and discuss how retromer contributes to a diverse set of physiological processes.

  18. Operon Gene Order Is Optimized for Ordered Protein Complex Assembly.

    PubMed

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2016-02-02

    The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.

  19. Mass Spectrometry of Protein Complexes: From Origins to Applications

    NASA Astrophysics Data System (ADS)

    Mehmood, Shahid; Allison, Timothy M.; Robinson, Carol V.

    2015-04-01

    Now routine is the ability to investigate soluble and membrane protein complexes in the gas phase of a mass spectrometer while preserving folded structure and ligand-binding properties. Several recent transformative developments have occurred to arrive at this point. These include advances in mass spectrometry instrumentation, particularly with respect to resolution; the ability to study intact membrane protein complexes released from detergent micelles; and the use of protein unfolding in the gas phase to obtain stability parameters. Together, these discoveries are providing unprecedented information on the compositional heterogeneity of biomacromolecules, the unfolding trajectories of multidomain proteins, and the stability imparted by ligand binding to both soluble and membrane-embedded protein complexes. We review these recent breakthroughs, highlighting the challenges that had to be overcome and the physicochemical insight that can now be gained from studying proteins and their assemblies in the gas phase.

  20. Isolation of lactic acid bacteria bound to the porcine intestinal mucosa and an analysis of their moonlighting adhesins

    PubMed Central

    KINOSHITA, Hideki; OHUCHI, Satoko; ARAKAWA, Kensuke; WATANABE, Masamichi; KITAZAWA, Haruki; SAITO, Tadao

    2016-01-01

    The adhesion of lactic acid bacteria (LAB) to the intestinal mucosa is one of the criteria in selecting for probiotics. Eighteen LAB were isolated from porcine intestinal mucin (PIM): ten strains of Lactobacillus, six strains of Weissella, and two strains of Streptococcus. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for phosphate-buffered saline (PBS) extracts from the LAB, many bands were detected in half of the samples, while a few and/or no clear bands were detected in the other half. All six of the selected LAB showed adhesion to PIM. L. johnsonii MYU 214 and MYU 221 showed adhesion at more than 10%. W. viridescens MYU 208, L. reuteri MYU 213, L. mucosae MYU 225, and L. agilis MYU 227 showed medium levels of adhesion at 5.9–8.3%. In a comprehensive analysis for the adhesins in the PBS extracts using a receptor overlay analysis, many moonlighting proteins were detected and identified as candidates for adhesins: GroEL, enolase, and elongation factor Tu in MYU 208; peptidase C1, enolase, formyl-CoA transferase, phosphoglyceromutase, triosephosphate isomerase, and phosphofructokinase in MYU 221; and DnaK, enolase, and phosphoglycerate kinase in MYU 227. These proteins in the PBS extracts, which included such things as molecular chaperones and glycolytic enzymes, may play important roles as adhesins. PMID:27867805

  1. Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC).

    PubMed

    Berger, Cedric N; Billker, Oliver; Meyer, Thomas F; Servin, Alain L; Kansau, Imad

    2004-05-01

    Little is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins. These adhesins recognize as receptors the GPI-anchored proteins CD55 (decay-accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA). CD66e is a member of the CEA-related cell adhesion molecules (CEACAM) family, comprising seven members. We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM-expressing CHO and HeLa cells. The results demonstrate that only E. coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr-I, including Dr, F1845 and AfaE-III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6. Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr-I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6. In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recruited around bacteria in HeLa cells. The recruited CEACAM1, CEA and CEACAM6 around adhering bacteria resist totally or in part a detergent extraction, whereas the recruited CEACAM3 does not. Finally, the results show that recognition of CEA and CEACAM6, but not CEACAM1, is accompanied by tight attachment to bacteria of cell surface microvilli-like extensions, which are elongated. Moreover, recognition of CEA is accompanied by an activation of the Rho GTPase Cdc42 and by a phosphorylation of ERM, which in turn elicit the observed cell surface microvilli-like extensions.

  2. Pseudomonas aeruginosa Minor Pilins Prime Type IVa Pilus Assembly and Promote Surface Display of the PilY1 Adhesin*

    PubMed Central

    Nguyen, Ylan; Sugiman-Marangos, Seiji; Harvey, Hanjeong; Bell, Stephanie D.; Charlton, Carmen L.; Junop, Murray S.; Burrows, Lori L.

    2015-01-01

    Type IV pili (T4P) contain hundreds of major subunits, but minor subunits are also required for assembly and function. Here we show that Pseudomonas aeruginosa minor pilins prime pilus assembly and traffic the pilus-associated adhesin and anti-retraction protein, PilY1, to the cell surface. PilV, PilW, and PilX require PilY1 for inclusion in surface pili and vice versa, suggestive of complex formation. PilE requires PilVWXY1 for inclusion, suggesting that it binds a novel interface created by two or more components. FimU is incorporated independently of the others and is proposed to couple the putative minor pilin-PilY1 complex to the major subunit. The production of small amounts of T4P by a mutant lacking the minor pilin operon was traced to expression of minor pseudopilins from the P. aeruginosa type II secretion (T2S) system, showing that under retraction-deficient conditions, T2S minor subunits can prime T4P assembly. Deletion of all minor subunits abrogated pilus assembly. In a strain lacking the minor pseudopilins, PilVWXY1 and either FimU or PilE comprised the minimal set of components required for pilus assembly. Supporting functional conservation of T2S and T4P minor components, our 1.4 Å crystal structure of FimU revealed striking architectural similarity to its T2S ortholog GspH, despite minimal sequence identity. We propose that PilVWXY1 form a priming complex for assembly and that PilE and FimU together stably couple the complex to the major subunit. Trafficking of the anti-retraction factor PilY1 to the cell surface allows for production of pili of sufficient length to support adherence and motility. PMID:25389296

  3. Computational approaches for detecting protein complexes from protein interaction networks: a survey

    PubMed Central

    2010-01-01

    Background Most proteins form macromolecular complexes to perform their biological functions. However, experimentally determined protein complex data, especially of those involving more than two protein partners, are relatively limited in the current state-of-the-art high-throughput experimental techniques. Nevertheless, many techniques (such as yeast-two-hybrid) have enabled systematic screening of pairwise protein-protein interactions en masse. Thus computational approaches for detecting protein complexes from protein interaction data are useful complements to the limited experimental methods. They can be used together with the experimental methods for mapping the interactions of proteins to understand how different proteins are organized into higher-level substructures to perform various cellular functions. Results Given the abundance of pairwise protein interaction data from high-throughput genome-wide experimental screenings, a protein interaction network can be constructed from protein interaction data by considering individual proteins as the nodes, and the existence of a physical interaction between a pair of proteins as a link. This binary protein interaction graph can then be used for detecting protein complexes using graph clustering techniques. In this paper, we review and evaluate the state-of-the-art techniques for computational detection of protein complexes, and discuss some promising research directions in this field. Conclusions Experimental results with yeast protein interaction data show that the interaction subgraphs discovered by various computational methods matched well with actual protein complexes. In addition, the computational approaches have also improved in performance over the years. Further improvements could be achieved if the quality of the underlying protein interaction data can be considered adequately to minimize the undesirable effects from the irrelevant and noisy sources, and the various biological evidences can be better

  4. Strategies for crystallization of large membrane protein complexes

    NASA Astrophysics Data System (ADS)

    Yoshikawa, Shinya; Shinzawa-Itoh, Kyoko; Ueda, Hidefumi; Tsukihara, Tomitake; Fukumoto, Yoshihisa; Kubota, Tomomi; Kawamoto, Masahide; Fukuyama, Keiichi; Matsubara, Hiroshi

    1992-08-01

    Crystalline cytochrome c oxidase and ubiquinol: cytochrome c oxidoreductase which diffracted X-rays at 7-8A˚resolution were obtained from bovine heart mitochondria. The methods for the purification and crystallization of these enzymes indicate that large membrane protein complexes are easier to purify and crystallize than smaller homologous membrane protein complexes, because the former have more hydrophilic surface than the latter. Bulky and polydispersed detergents such as Brij-35 and Tween 20 attached to the isolated complex are not always obstructive to crystallization if they are effective for stabilizing the complexes.

  5. Sizing Large Proteins and Protein Complexes by Electrospray Ionization Mass Spectrometry and Ion Mobility

    PubMed Central

    Kaddis, Catherine S.; Lomeli, Shirley H.; Yin, Sheng; Berhane, Beniam; Apostol, Marcin I.; Kickhoefer, Valerie A.; Rome, Leonard H.; Loo, Joseph A.

    2009-01-01

    Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method termed GEMMA (Gas-Phase Electrophoretic Mobility Molecular Analysis), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared to other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and x-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm3. Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water. PMID:17434746

  6. Nanoparticle-protein complexes mimicking corona formation in ocular environment.

    PubMed

    Jo, Dong Hyun; Kim, Jin Hyoung; Son, Jin Gyeong; Dan, Ki Soon; Song, Sang Hoon; Lee, Tae Geol; Kim, Jeong Hun

    2016-12-01

    Nanoparticles adsorb biomolecules to form corona upon entering the biological environment. In this study, tissue-specific corona formation is provided as a way of controlling protein interaction with nanoparticles in vivo. In the vitreous, the composition of the corona was determined by the electrostatic and hydrophobic properties of the associated proteins, regardless of the material (gold and silica) or size (20- and 100-nm diameter) of the nanoparticles. To control protein adsorption, we pre-incubate 20-nm gold nanoparticles with 5 selectively enriched proteins from the corona, formed in the vitreous, to produce nanoparticle-protein complexes. Compared to bare nanoparticles, nanoparticle-protein complexes demonstrate improved binding to vascular endothelial growth factor (VEGF) in the vitreous. Furthermore, nanoparticle-protein complexes retain in vitro anti-angiogenic properties of bare nanoparticles. In particular, priming the nanoparticles (gold and silica) with tissue-specific corona proteins allows nanoparticle-protein complexes to exert better in vivo therapeutic effects by higher binding to VEGF than bare nanoparticles. These results suggest that controlled corona formation that mimics in vivo processes may be useful in the therapeutic use of nanomaterials in local environment.

  7. Unwinding protein complexes in ALTernative telomere maintenance.

    PubMed

    Bhattacharyya, Saumitri; Sandy, April; Groden, Joanna

    2010-01-01

    Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA-binding proteins and a number of three dimensional nucleic acid structures including G-quartets and D-loops. A number of studies suggest that the BLM and WRN recQ-like helicases play important roles in recombination-mediated mechanisms of telomere elongation or Alternative Lengthening of Telomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT-associated nuclear bodies in telomerase-negative immortalized cell lines and interact with the telomere-specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double-strand break repair processes such as non-homologous end joining, homologous recombination-mediated repair, resolution of stalled replication forks and synthesis-dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ-like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec-Q-like helicases and their role in telomere elongation during ALT processes.

  8. From quantitative protein complex analysis to disease mechanism.

    PubMed

    Texier, Y; Kinkl, N; Boldt, K; Ueffing, M

    2012-12-15

    Interest in the field of cilia biology and cilia-associated diseases - ciliopathies - has strongly increased over the last few years. Proteomic technologies, especially protein complex analysis by affinity purification-based methods, have been used to decipher various basic but also disease-associated mechanisms. This review focusses on some selected recent studies using affinity purification-based protein complex analysis, thereby exemplifying the great possibilities this technology offers.

  9. Dystrophin complex functions as a scaffold for signalling proteins.

    PubMed

    Constantin, Bruno

    2014-02-01

    Dystrophin is a 427kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part

  10. Mass Spectrometry of Protein-Ligand Complexes: Enhanced Gas Phase Stability of Ribonuclease-Nucleotide Complexes

    PubMed Central

    Yin, Sheng; Xie, Yongming; Loo, Joseph A.

    2008-01-01

    Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein-ligand complexes depends, however, on the stability of the complexes in the gas phase environment. Solution binding affinities may or may not be accurate predictors of their stability in vacuo. Complexes composed of cytidine nucleotides bound to ribonuclease A (RNase A) and ribonuclease S (RNase S) were detected by ESI-MS and were further analyzed by MS/MS. RNase A and RNase S share similar structures and biological activity. Subtilisin-cleavage of RNase A yields an S-peptide and an S-protein; the S-peptide and S-protein interact through hydrophobic interactions with a solution binding constant in the nanomolar range to generate an active RNase S. Cytidine nucleotides bind to the ribonucleases through electrostatic interactions with a solution binding constant in the micromolar range. Collisionally activated dissociation (CAD) of the 1:1 RNase A-CDP and CTP complexes yields cleavage of the covalent phosphate bonds of the nucleotide ligands, releasing CMP from the complex. CAD of the RNase S-CDP and CTP complexes dissociates the S-peptide from the remaining S-protein/nucleotide complex; further dissociation of the S-protein/nucleotide complex fragments a covalent phosphate bond of the nucleotide with subsequent release of CMP. Despite a solution binding constant favoring the S-protein/S-peptide complex, CDP/CTP remains electrostatically bound to the S-protein in the gas phase dissociation experiment. This study highlights the intrinsic stability of electrostatic interactions in the gas phase and the significant differences in solution and gas phase stabilities of noncovalent complexes that can result. PMID:18565758

  11. The dynamics and pH-dependence of Ag43 adhesins' self-association probed by atomic force spectroscopy

    NASA Astrophysics Data System (ADS)

    Jacquot, Adrien; Sakamoto, Chizuko; Razafitianamarahavo, Angelina; Caillet, Céline; Merlin, Jenny; Fahs, Ahmad; Ghigo, Jean-Marc; Duval, Jérôme F. L.; Beloin, Christophe; Francius, Grégory

    2014-10-01

    Self-associating auto-transporter (SAAT) adhesins are two-domain cell surface proteins involved in bacteria auto-aggregation and biofilm formation. Antigen 43 (Ag43) is a SAAT adhesin commonly found in Escherichia coli whose variant Ag43a has been shown to promote persistence of uropathogenic E. coli within the bladder. The recent resolution of the tri-dimensional structure of the 499 amino-acids' β-domain in Ag43a has shed light on the possible mechanism governing the self-recognition of SAAT adhesins, in particular the importance of trans-interactions between the L shaped β-helical scaffold of two α-domains of neighboring adhesins. In this study, we use single-molecule force spectroscopy (SMFS) and dynamic force spectroscopy (DFS) to unravel the dynamics of Ag43-self association under various pH and molecular elongation rate conditions that mimic the situations encountered by E. coli in its natural environment. Results evidenced an important stretchability of Ag43α with unfolding of sub-domains leading to molecular extension as long as 150 nm. Nanomechanical analysis of molecular stretching data suggested that self-association of Ag43 can lead to the formation of dimers and tetramers driven by rapid and weak cis- as well as slow but strong trans-interaction forces with a magnitude as large as 100-250 pN. The dynamics of cis- and trans-interactions were demonstrated to be strongly influenced by pH and applied shear force, thus suggesting that environmental conditions can modulate Ag43-mediated aggregation of bacteria at the molecular level.Self-associating auto-transporter (SAAT) adhesins are two-domain cell surface proteins involved in bacteria auto-aggregation and biofilm formation. Antigen 43 (Ag43) is a SAAT adhesin commonly found in Escherichia coli whose variant Ag43a has been shown to promote persistence of uropathogenic E. coli within the bladder. The recent resolution of the tri-dimensional structure of the 499 amino-acids' β-domain in Ag43a has shed

  12. Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

    SciTech Connect

    Bordner, Andrew J.; Gorin, Andrey A.

    2008-05-12

    Here, protein-protein interactions are ubiquitous and essential for cellular processes. High-resolution X-ray crystallographic structures of protein complexes can elucidate the details of their function and provide a basis for many computational and experimental approaches. Here we demonstrate that existing annotations of protein complexes, including those provided by the Protein Data Bank (PDB) itself, contain a significant fraction of incorrect annotations. Results: We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1) comprehensively collecting all protein-protein interfaces; (2) clustering similar protein-protein interfaces together; (3) estimating the probability that each cluster is relevant based on a diverse set of properties; and (4) finally combining these scores for each entry in order to predict the complex structure. Unlike previous annotation methods, consistent prediction of complexes with identical or almost identical protein content is insured. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions.

  13. Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

    DOE PAGES

    Bordner, Andrew J.; Gorin, Andrey A.

    2008-05-12

    Here, protein-protein interactions are ubiquitous and essential for cellular processes. High-resolution X-ray crystallographic structures of protein complexes can elucidate the details of their function and provide a basis for many computational and experimental approaches. Here we demonstrate that existing annotations of protein complexes, including those provided by the Protein Data Bank (PDB) itself, contain a significant fraction of incorrect annotations. Results: We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1) comprehensively collecting all protein-protein interfaces; (2) clustering similar protein-protein interfaces together; (3) estimating the probability that each cluster ismore » relevant based on a diverse set of properties; and (4) finally combining these scores for each entry in order to predict the complex structure. Unlike previous annotation methods, consistent prediction of complexes with identical or almost identical protein content is insured. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions.« less

  14. Role of Staphylococcus aureus surface adhesins in orthopaedic device infections: are results model-dependent?

    PubMed

    Darouiche, R O; Landon, G C; Patti, J M; Nguyen, L L; Fernau, R C; McDevitt, D; Greene, C; Foster, T; Klima, M

    1997-01-01

    Bacterial colonisation of prosthetic material can lead to clinical infection or implant failure, or both, often requiring removal of the device. Adherence of Staphylococcus aureus to bioprosthetic materials is mediated by adhesins belonging to the MSCRAMM (microbial surface components recognising adhesive matrix molecules) family of microbial cell surface proteins. The objective of this study was to compare the virulence of a mutant strain of S. aureus Newman that possesses all three fibrinogen-, fibronectin- and collagen-binding MSCRAMMs (MSCRAMM-positive strain) with that of a mutant strain that lacks all three types of MSCRAMMs (MSCRAMM-negative strain) in a rabbit model of orthopaedic device-related infection. After a hole was drilled into the knee joint of each animal, a group of 10 rabbits was inoculated with the MSCRAMM-positive strain and another group of 10 rabbits received the MSCRAMM-negative strain. A stainless steel screw was then placed into the drilled hole. Two weeks later, the rabbits were killed and serum samples, bone tissue and implants were harvested for bacteriological and histopathological evaluation. No significant difference in infection rates was demonstrated between the two groups. The ability to delineate the role of S. aureus surface adhesins in causing orthopaedic device-related infection could be model-dependent.

  15. Adenosine deaminase complexing protein in cancer studies.

    PubMed

    Ten Kate, J; Dinjens, W N; Meera Khan, P; Bosman, F T

    1986-01-01

    ADCP is a dimeric glycoprotein of about 200KD, for which the physiological role is still obscure. This protein occurs mainly in a membrane bound form in various human tissues. In this paper we review the current literature on ADCP in cancer studies. Soluble ADCP was described to be consistently decreased or absent in cancers of lung, liver, kidney and colon. These findings could not be confirmed by immunohistochemical and quantitative biochemical studies in a series of colorectal-, prostatic-, and renal carcinomas. Only in a third of these tumors a decrease could be demonstrated, whereas in the other cases unaltered or even increased amounts were observed. However, in virally transformed human fibroblasts a consistent decrease or complete absence of ADCP was seen, while primary fibroblasts were found to contain high amounts of this protein. Recently, the use of ADCP as a differentiation marker in colonic cancer has been advocated. Furthermore the presence of ADCP in the serum of renal adenocarcinoma patients was found to be indicative of a better chance of five year survival. These studies suggest that ADCP may be a differentiation marker useful for immunohistochemical characterization of colonic and renal carcinomas as well as a serum marker useful for follow-up studies of these types of cancer, analogous to CEA. Finally, ADCP has been found to be selectively expressed by certain T-cell subsets and henceforth may be useful in the studies on leukemias.

  16. Biochemical isolation of Argonaute protein complexes by Ago-APP

    PubMed Central

    Hauptmann, Judith; Schraivogel, Daniel; Bruckmann, Astrid; Manickavel, Sudhir; Jakob, Leonhard; Eichner, Norbert; Pfaff, Janina; Urban, Marc; Sprunck, Stefanie; Hafner, Markus; Tuschl, Thomas; Deutzmann, Rainer; Meister, Gunter

    2015-01-01

    During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells. PMID:26351695

  17. Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings

    PubMed Central

    Brzozowska, Ewa; Koba, Marcin; Śmietana, Mateusz; Górska, Sabina; Janik, Monika; Gamian, Andrzej; Bock, Wojtek J.

    2016-01-01

    This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests – ELISA and BIAcore – the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria. PMID:27231592

  18. The Haemophilus ducreyi trimeric autotransporter adhesin DsrA protects against an experimental infection in the swine model of chancroid.

    PubMed

    Fusco, William G; Choudhary, Neelima R; Routh, Patty A; Ventevogel, Melissa S; Smith, Valerie A; Koch, Gary G; Almond, Glen W; Orndorff, Paul E; Sempowski, Gregory D; Leduc, Isabelle

    2014-06-24

    Adherence of pathogens to cellular targets is required to initiate most infections. Defining strategies that interfere with adhesion is therefore important for the development of preventative measures against infectious diseases. As an adhesin to host extracellular matrix proteins and human keratinocytes, the trimeric autotransporter adhesin DsrA, a proven virulence factor of the Gram-negative bacterium Haemophilus ducreyi, is a potential target for vaccine development. A recombinant form of the N-terminal passenger domain of DsrA from H. ducreyi class I strain 35000HP, termed rNT-DsrAI, was tested as a vaccine immunogen in the experimental swine model of H. ducreyi infection. Viable homologous H. ducreyi was not recovered from any animal receiving four doses of rNT-DsrAI administered with Freund's adjuvant at two-week intervals. Control pigs receiving adjuvant only were all infected. All animals receiving the rNT-DsrAI vaccine developed antibody endpoint titers between 3.5 and 5 logs. All rNT-DsrAI antisera bound the surface of the two H. ducreyi strains used to challenge immunized pigs. Purified anti-rNT-DsrAI IgG partially blocked binding of fibrinogen at the surface of viable H. ducreyi. Overall, immunization with the passenger domain of the trimeric autotransporter adhesin DsrA accelerated clearance of H. ducreyi in experimental lesions, possibly by interfering with fibrinogen binding.

  19. Luminescent iridium(III) complexes as novel protein staining agents.

    PubMed

    Jia, Junli; Fei, Hao; Zhou, Ming

    2012-05-01

    This article reports a new class of luminescent metal complexes, biscyclometalated iridium(III) complexes with an ancillary bathophenanthroline disulfonate ligand, for staining protein bands that are separated by electrophoresis. The performances of these novel staining agents have been studied in comparison with tris(bathophenanthroline disulfonate) ruthenium(II) tetrasodium salt (i.e. RuBPS) using a commercially available imaging system. The staining agents showed different limits of detection, linear dynamic ranges, and protein-to-protein variations. The overall performances of all three stains were found to be better than or equivalent to RuBPS under the experimental conditions.

  20. Protein Complex Production from the Drug Discovery Standpoint.

    PubMed

    Moarefi, Ismail

    2016-01-01

    Small molecule drug discovery critically depends on the availability of meaningful in vitro assays to guide medicinal chemistry programs that are aimed at optimizing drug potency and selectivity. As it becomes increasingly evident, most disease relevant drug targets do not act as a single protein. In the body, they are instead generally found in complex with protein cofactors that are highly relevant for their correct function and regulation. This review highlights selected examples of the increasing trend to use biologically relevant protein complexes for rational drug discovery to reduce costly late phase attritions due to lack of efficacy or toxicity.

  1. Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

    PubMed Central

    Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.

    2013-01-01

    Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423

  2. Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

    PubMed Central

    Miczak, A; Kaberdin, V R; Wei, C L; Lin-Chao, S

    1996-01-01

    The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed. Images Fig. 1 Fig. 2 PMID:8632981

  3. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes

    PubMed Central

    Luo, Jiawei; Qi, Yi

    2015-01-01

    Background Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins. Method In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC), based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID), of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification. Results Experimental results based on three different PPI(protein-protein interaction) networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC). Conclusions LIDC is more effective for the prediction of essential proteins than other recently developed methods. PMID:26125187

  4. Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes

    SciTech Connect

    Martinovic, Suzana; Pasa-Tolic, Liljiana; Smith, Richard D.

    2004-10-01

    Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of non-covalent complexes on the intact complex level.

  5. Global landscape of HIV-human protein complexes.

    PubMed

    Jäger, Stefanie; Cimermancic, Peter; Gulbahce, Natali; Johnson, Jeffrey R; McGovern, Kathryn E; Clarke, Starlynn C; Shales, Michael; Mercenne, Gaelle; Pache, Lars; Li, Kathy; Hernandez, Hilda; Jang, Gwendolyn M; Roth, Shoshannah L; Akiva, Eyal; Marlett, John; Stephens, Melanie; D'Orso, Iván; Fernandes, Jason; Fahey, Marie; Mahon, Cathal; O'Donoghue, Anthony J; Todorovic, Aleksandar; Morris, John H; Maltby, David A; Alber, Tom; Cagney, Gerard; Bushman, Frederic D; Young, John A; Chanda, Sumit K; Sundquist, Wesley I; Kortemme, Tanja; Hernandez, Ryan D; Craik, Charles S; Burlingame, Alma; Sali, Andrej; Frankel, Alan D; Krogan, Nevan J

    2011-12-21

    Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with ∼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.

  6. Antibodies to the HMW1/HMW2 and Hia Adhesins of Nontypeable Haemophilus influenzae Mediate Broad-Based Opsonophagocytic Killing of Homologous and Heterologous Strains

    PubMed Central

    Winter, Linda E.

    2014-01-01

    The HMW1/HMW2 and Hia proteins are highly immunogenic surface adhesins of nontypeable Haemophilus influenzae (NTHi). Approximately 75% of NTHi strains express HMW1/HMW2 adhesins, and most of the remaining 25% express an Hia adhesin. Our objective in this study was to assess the ability of antisera raised against purified HMW1/HMW2 proteins or recombinant Hia proteins to mediate opsonophagocytic killing of a large panel of unrelated NTHi strains. Native HMW1/HMW2 proteins were purified from three HMW1/HMW2-expressing NTHi strains. Recombinant fusion proteins expressing surface-exposed segments of either of two prototype Hia proteins were purified from Escherichia coli transformants. Immune sera raised in guinea pigs were assessed for their ability to mediate killing of NTHi in an opsonophagocytic assay with the HL-60 phagocytic cell line. The three HMW1/HMW2 antisera mediated killing of 22 of 65, 43 of 65, and 28 of 65 unrelated HMW1/HMW2-expressing NTHi strains, respectively. As a group, the three sera mediated killing of 48 of 65 HMW1/HMW2-expressing strains. The two Hia immune sera mediated killing of 12 of 24 and 13 of 24 unrelated Hia-expressing NTHi strains, respectively. Together, they mediated killing of 15 of 24 Hia-expressing strains. Neither the HMW1/HMW2 nor the Hia antisera mediated killing of NTHi expressing the alternative adhesin type. Antibodies directed against native HMW1/HMW2 proteins and recombinant Hia proteins are capable of mediating broad-based opsonophagocytic killing of homologous and heterologous NTHi strains. A vaccine formulated with a limited number of HMW1/HMW2 and Hia proteins might provide protection against disease caused by most NTHi strains. PMID:24574538

  7. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  8. That's my story, and I'm sticking to it—an update on B. burgdorferi adhesins

    PubMed Central

    Brissette, Catherine A.; Gaultney, Robert A.

    2014-01-01

    Adhesion is the initial event in the establishment of any infection. Borrelia burgdorferi, the etiological agent of Lyme disease, possesses myriad proteins termed adhesins that facilitate contact with its vertebrate hosts. B. burgdorferi adheres to host tissues through interactions with host cells and extracellular matrix, as well as other molecules present in serum and extracellular fluids. These interactions, both general and specific, are critical in the establishment of infection. Modulation of borrelial adhesion to host tissues affects the microorganisms's ability to colonize, disseminate, and persist. In this review, we update the current knowledge on structure, function, and role in pathogenesis of these “sticky” B. burgdorferi infection-associated proteins. PMID:24772392

  9. Burkholderia cenocepacia K56-2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation.

    PubMed

    Mil-Homens, Dalila; Pinto, Sandra N; Matos, Rute G; Arraiano, Cecília; Fialho, Arsenio M

    2017-04-01

    Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF-associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56-2, is a tumor necrosis factor receptor 1-interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL-8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence.

  10. Chimeric Protein Complexes in Hybrid Species Generate Novel Phenotypes

    PubMed Central

    Piatkowska, Elzbieta M.; Naseeb, Samina; Knight, David; Delneri, Daniela

    2013-01-01

    Hybridization between species is an important mechanism for the origin of novel lineages and adaptation to new environments. Increased allelic variation and modification of the transcriptional network are the two recognized forces currently deemed to be responsible for the phenotypic properties seen in hybrids. However, since the majority of the biological functions in a cell are carried out by protein complexes, inter-specific protein assemblies therefore represent another important source of natural variation upon which evolutionary forces can act. Here we studied the composition of six protein complexes in two different Saccharomyces “sensu stricto” hybrids, to understand whether chimeric interactions can be freely formed in the cell in spite of species-specific co-evolutionary forces, and whether the different types of complexes cause a change in hybrid fitness. The protein assemblies were isolated from the hybrids via affinity chromatography and identified via mass spectrometry. We found evidence of spontaneous chimericity for four of the six protein assemblies tested and we showed that different types of complexes can cause a variety of phenotypes in selected environments. In the case of TRP2/TRP3 complex, the effect of such chimeric formation resulted in the fitness advantage of the hybrid in an environment lacking tryptophan, while only one type of parental combination of the MBF complex allowed the hybrid to grow under respiratory conditions. These phenotypes were dependent on both genetic and environmental backgrounds. This study provides empirical evidence that chimeric protein complexes can freely assemble in cells and reveals a new mechanism to generate phenotypic novelty and plasticity in hybrids to complement the genomic innovation resulting from gene duplication. The ability to exchange orthologous members has also important implications for the adaptation and subsequent genome evolution of the hybrids in terms of pattern of gene loss. PMID

  11. The Three-dimensional Structure of the Extracellular Adhesion Domain of the Sialic Acid-binding Adhesin SabA from Helicobacter pylori

    PubMed Central

    Pang, Siew Siew; Nguyen, Stanley Thai Son; Perry, Andrew J.; Day, Christopher J.; Panjikar, Santosh; Tiralongo, Joe; Whisstock, James C.; Kwok, Terry

    2014-01-01

    The gastric pathogen Helicobacter pylori is a major cause of acute chronic gastritis and the development of stomach and duodenal ulcers. Chronic infection furthermore predisposes to the development of gastric cancer. Crucial to H. pylori survival within the hostile environment of the digestive system are the adhesins SabA and BabA; these molecules belong to the same protein family and permit the bacteria to bind tightly to sugar moieties LewisB and sialyl-LewisX, respectively, on the surface of epithelial cells lining the stomach and duodenum. To date, no representative SabA/BabA structure has been determined, hampering the development of strategies to eliminate persistent H. pylori infections that fail to respond to conventional therapy. Here, using x-ray crystallography, we show that the soluble extracellular adhesin domain of SabA shares distant similarity to the tetratricopeptide repeat fold family. The molecule broadly resembles a golf putter in shape, with the head region featuring a large cavity surrounded by loops that vary in sequence between different H. pylori strains. The N-terminal and C-terminal helices protrude at right angles from the head domain and together form a shaft that connects to a predicted outer membrane protein-like β-barrel trans-membrane domain. Using surface plasmon resonance, we were able to detect binding of the SabA adhesin domain to sialyl-LewisX and LewisX but not to LewisA, LewisB, or LewisY. Substitution of the highly conserved glutamine residue 159 in the predicted ligand-binding pocket abrogates the binding of the SabA adhesin domain to sialyl-LewisX and LewisX. Taken together, these data suggest that the adhesin domain of SabA is sufficient in isolation for specific ligand binding. PMID:24375407

  12. Machine Learning Approaches for Predicting Protein Complex Similarity.

    PubMed

    Farhoodi, Roshanak; Akbal-Delibas, Bahar; Haspel, Nurit

    2017-01-01

    Discriminating native-like structures from false positives with high accuracy is one of the biggest challenges in protein-protein docking. While there is an agreement on the existence of a relationship between various favorable intermolecular interactions (e.g., Van der Waals, electrostatic, and desolvation forces) and the similarity of a conformation to its native structure, the precise nature of this relationship is not known. Existing protein-protein docking methods typically formulate this relationship as a weighted sum of selected terms and calibrate their weights by using a training set to evaluate and rank candidate complexes. Despite improvements in the predictive power of recent docking methods, producing a large number of false positives by even state-of-the-art methods often leads to failure in predicting the correct binding of many complexes. With the aid of machine learning methods, we tested several approaches that not only rank candidate structures relative to each other but also predict how similar each candidate is to the native conformation. We trained a two-layer neural network, a multilayer neural network, and a network of Restricted Boltzmann Machines against extensive data sets of unbound complexes generated by RosettaDock and PyDock. We validated these methods with a set of refinement candidate structures. We were able to predict the root mean squared deviations (RMSDs) of protein complexes with a very small, often less than 1.5 Å, error margin when trained with structures that have RMSD values of up to 7 Å. In our most recent experiments with the protein samples having RMSD values up to 27 Å, the average prediction error was still relatively small, attesting to the potential of our approach in predicting the correct binding of protein-protein complexes.

  13. β-Barrel membrane protein assembly by the Bam complex.

    PubMed

    Hagan, Christine L; Silhavy, Thomas J; Kahne, Daniel

    2011-01-01

    β-barrel membrane proteins perform important functions in the outer membranes (OMs) of Gram-negative bacteria and of the mitochondria and chloroplasts of eukaryotes. The protein complexes that assemble these proteins in their respective membranes have been identified and shown to contain a component that has been conserved from bacteria to humans. β-barrel proteins are handled differently from α-helical membrane proteins in the cell in order to efficiently transport them to their final locations in unfolded but folding-competent states. The mechanism by which the assembly complex then binds, folds, and inserts β-barrels into the membrane is not well understood, but recent structural, biochemical, and genetic studies have begun to elucidate elements of how the complex provides a facilitated pathway for β-barrel assembly. Ultimately, studies of the mechanism of β-barrel assembly and comparison to the better-understood process of α-helical membrane protein assembly will reveal whether there are general principles that guide the folding and insertion of all membrane proteins.

  14. SgrA, a nidogen-binding LPXTG surface adhesin implicated in biofilm formation, and EcbA, a collagen binding MSCRAMM, are two novel adhesins of hospital-acquired Enterococcus faecium.

    PubMed

    Hendrickx, Antoni P A; van Luit-Asbroek, Miranda; Schapendonk, Claudia M E; van Wamel, Willem J B; Braat, Johanna C; Wijnands, Lucas M; Bonten, Marc J M; Willems, Rob J L

    2009-11-01

    Hospital-acquired Enterococcus faecium isolates responsible for nosocomial outbreaks and invasive infections are enriched in the orf2351 and orf2430 genes, encoding the SgrA and EcbA LPXTG-like cell wall-anchored proteins, respectively. These two surface proteins were characterized to gain insight into their function, since they may have favored the rapid emergence of this nosocomial pathogen. We are the first to identify a surface adhesin among bacteria (SgrA) that binds to the extracellular matrix molecules nidogen 1 and nidogen 2, which are constituents of the basal lamina. EcbA is a novel E. faecium MSCRAMM (microbial surface component recognizing adhesive matrix molecules) that binds to collagen type V. In addition, both SgrA and EcbA bound to fibrinogen; however, SgrA targeted the alpha and beta chains, whereas EcbA bound to the gamma chain of fibrinogen. An E. faecium sgrA insertion mutant displayed reduced binding to both nidogens and fibrinogen. SgrA did not mediate binding of E. faecium cells to biotic materials, such as human intestinal epithelial cells, human bladder cells, and kidney cells, while this LPXTG surface adhesin is implicated in E. faecium biofilm formation. The acm and scm genes, encoding two other E. faecium MSCRAMMs, were expressed at the mRNA level together with sgrA during all phases of growth, whereas ecbA was expressed only in exponential and late exponential phase, suggesting orchestrated expression of these adhesins. Expression of these surface proteins, which bind to extracellular matrix proteins and are involved in biofilm formation (SgrA), may contribute to the pathogenesis of hospital-acquired E. faecium infections.

  15. Characterization of invasive Neisseria meningitidis from Atlantic Canada, 2009 to 2013: With special reference to the nonpolysaccharide vaccine targets (PorA, factor H binding protein, Neisseria heparin-binding antigen and Neisseria adhesin A)

    PubMed Central

    Tsang, Raymond SW; Law, Dennis KS; Gad, Rita R; Mailman, Tim; German, Gregory; Needle, Robert

    2015-01-01

    BACKGROUND: Serogroup B Neisseria meningitidis (MenB) has always been a major cause of invasive meningococcal disease (IMD) in Canada. With the successful implementation of a meningitis C conjugate vaccine, the majority of IMD in Canada is now caused by MenB. OBJECTIVE: To investigate IMD case isolates in Atlantic Canada from 2009 to 2013. Data were analyzed to determine the potential coverage of the newly licensed MenB vaccine. METHODS: Serogroup, serotype and serosubtype antigens were determined from IMD case isolates. Clonal analysis was performed using multilocus sequence typing. The protein-based vaccine antigen genes were sequenced and the predicted peptides were investigated. RESULTS: The majority of the IMD isolates were MenB (82.5%, 33 of 40) and, in particular, sequence type (ST)-154 B:4:P1.4 was responsible for 47.5% (19 of 40) of all IMD case isolates in Atlantic Canada. Isolates of this clone expressed the PorA antigen P1.4 and possessed the nhba genes encoding for Neisseria heparin-binding antigen peptide 2, which together matched exactly with two of the four components of the new four-component meningococcal B vaccine. Nineteen MenB isolates had two antigenic matches, another five MenB and one meningitis Y isolate had one antigenic match. This provided 75.8% (25 of 33) potential coverage for MenB, or a 62.5% (25 of 40) overall potential coverage for IMD. CONCLUSION: From 2009 to 2013, IMD in Atlantic Canada was mainly caused by MenB and, in particular, the B:4:P1.4 ST-154 clone, which accounted for 47.5% of all IMD case isolates. The new four-component meningococcal B vaccine appeared to offer adequate coverage against MenB in Atlantic Canada. PMID:26744586

  16. Protein-complex structure completion using IPCAS (Iterative Protein Crystal structure Automatic Solution).

    PubMed

    Zhang, Weizhe; Zhang, Hongmin; Zhang, Tao; Fan, Haifu; Hao, Quan

    2015-07-01

    Protein complexes are essential components in many cellular processes. In this study, a procedure to determine the protein-complex structure from a partial molecular-replacement (MR) solution is demonstrated using a direct-method-aided dual-space iterative phasing and model-building program suite, IPCAS (Iterative Protein Crystal structure Automatic Solution). The IPCAS iteration procedure involves (i) real-space model building and refinement, (ii) direct-method-aided reciprocal-space phase refinement and (iii) phase improvement through density modification. The procedure has been tested with four protein complexes, including two previously unknown structures. It was possible to use IPCAS to build the whole complex structure from one or less than one subunit once the molecular-replacement method was able to give a partial solution. In the most challenging case, IPCAS was able to extend to the full length starting from less than 30% of the complex structure, while conventional model-building procedures were unsuccessful.

  17. Emergence of Complexity in Protein Functions and Metabolic Networks

    NASA Technical Reports Server (NTRS)

    Pohorille, Andzej

    2009-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis of very large libraries of random amino acid sequences and subsequently subjecting them to in vitro evolution. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions, important clues have been uncovered. Considerable progress has been also achieved in understanding the origins of membrane proteins. We will address this issue in the example of ion channels - proteins that mediate transport of ions across cell walls. Remarkably, despite overall complexity of these proteins in contemporary cells, their structural motifs are quite simple, with -helices being most common. By combining results of experimental and computer simulation studies on synthetic models and simple, natural channels, I will show that, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during

  18. Subcellular localization of RNA degrading proteins and protein complexes in prokaryotes.

    PubMed

    Evguenieva-Hackenberg, Elena; Roppelt, Verena; Lassek, Christian; Klug, Gabriele

    2011-01-01

    The archaeal exosome is a prokaryotic protein complex with RNA processing and degrading activities. Recently it was shown that the exosome is localized at the periphery of the cell in the thermoacidophilic archaeon Sulfolobus solfataricus. This localization is most likely mediated by the archaeal DnaG protein and depends on (direct or indirect) hydrophobic interactions with the membrane. A localization of RNA degrading proteins and protein complexes was also demonstrated in several bacteria. In bacteria a subcellular localization was also shown for substrates of these proteins and protein complexes, i.e. chromosomally encoded mRNAs and a small RNA. Thus, despite the missing compartmentalization, a spatial organization of RNA processing and degradation exists in prokaryotic cells. Recent data suggest that the spatial organization contributes to the temporal regulation of these processes.

  19. Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA.

    PubMed

    Fusco, William G; Choudhary, Neelima R; Stewart, Shelley M; Alam, S Munir; Sempowski, Gregory D; Elkins, Christopher; Leduc, Isabelle

    2015-04-01

    Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrA(I)) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine.

  20. 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

  1. 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.

  2. Synthetic RNA-protein complex shaped like an equilateral triangle

    NASA Astrophysics Data System (ADS)

    Ohno, Hirohisa; Kobayashi, Tetsuhiro; Kabata, Rinko; Endo, Kei; Iwasa, Takuma; Yoshimura, Shige H.; Takeyasu, Kunio; Inoue, Tan; Saito, Hirohide

    2011-02-01

    Synthetic nanostructures consisting of biomacromolecules such as nucleic acids have been constructed using bottom-up approaches. In particular, Watson-Crick base pairing has been used to construct a variety of two- and three-dimensional DNA nanostructures. Here, we show that RNA and the ribosomal protein L7Ae can form a nanostructure shaped like an equilateral triangle that consists of three proteins bound to an RNA scaffold. The construction of the complex relies on the proteins binding to kink-turn (K-turn) motifs in the RNA, which allows the RNA to bend by ~60° at three positions to form a triangle. Functional RNA-protein complexes constructed with this approach could have applications in nanomedicine and synthetic biology.

  3. Structural and evolutionary versatility in protein complexes with uneven stoichiometry.

    PubMed

    Marsh, Joseph A; Rees, Holly A; Ahnert, Sebastian E; Teichmann, Sarah A

    2015-03-16

    Proteins assemble into complexes with diverse quaternary structures. Although most heteromeric complexes of known structure have even stoichiometry, a significant minority have uneven stoichiometry--that is, differing numbers of each subunit type. To adopt this uneven stoichiometry, sequence-identical subunits must be asymmetric with respect to each other, forming different interactions within the complex. Here we first investigate the occurrence of uneven stoichiometry, demonstrating that it is common in vitro and is likely to be common in vivo. Next, we elucidate the structural determinants of uneven stoichiometry, identifying six different mechanisms by which it can be achieved. Finally, we study the frequency of uneven stoichiometry across evolution, observing a significant enrichment in bacteria compared with eukaryotes. We show that this arises due to a general increased tendency for bacterial proteins to self-assemble and form homomeric interactions, even within the context of a heteromeric complex.

  4. Molecular dynamics simulations of a membrane protein/amphipol complex.

    PubMed

    Perlmutter, Jason D; Popot, Jean-Luc; Sachs, Jonathan N

    2014-10-01

    Amphipathic polymers known as "amphipols" provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.

  5. Biclustering Protein Complex Interactions with a Biclique FindingAlgorithm

    SciTech Connect

    Ding, Chris; Zhang, Anne Ya; Holbrook, Stephen

    2006-12-01

    Biclustering has many applications in text mining, web clickstream mining, and bioinformatics. When data entries are binary, the tightest biclusters become bicliques. We propose a flexible and highly efficient algorithm to compute bicliques. We first generalize the Motzkin-Straus formalism for computing the maximal clique from L{sub 1} constraint to L{sub p} constraint, which enables us to provide a generalized Motzkin-Straus formalism for computing maximal-edge bicliques. By adjusting parameters, the algorithm can favor biclusters with more rows less columns, or vice verse, thus increasing the flexibility of the targeted biclusters. We then propose an algorithm to solve the generalized Motzkin-Straus optimization problem. The algorithm is provably convergent and has a computational complexity of O(|E|) where |E| is the number of edges. It relies on a matrix vector multiplication and runs efficiently on most current computer architectures. Using this algorithm, we bicluster the yeast protein complex interaction network. We find that biclustering protein complexes at the protein level does not clearly reflect the functional linkage among protein complexes in many cases, while biclustering at protein domain level can reveal many underlying linkages. We show several new biologically significant results.

  6. Architecture and function of IFT complex proteins in ciliogenesis.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Lorentzen, Esben

    2012-02-01

    Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT.

  7. Single-protein study of photoresistance of pigment-protein complex in lipid bilayer

    NASA Astrophysics Data System (ADS)

    Uchiyama, Daisuke; Hoshino, Hajime; Otomo, Kohei; Kato, Taro; Onda, Ken-ichi; Watanabe, Akira; Oikawa, Hiroyuki; Fujiyoshi, Satoru; Matsushita, Michio; Nango, Mamoru; Watanabe, Natsuko; Sumino, Ayumi; Dewa, Takehisa

    2011-07-01

    Photoresistance of a pigment-binding membrane protein, light-harvesting 2 (LH2) complex from the photosynthetic bacterium, Rhodopseudomonas acidophila, was investigated by fluorescence of single LH2 complexes at a temperature of 296 K. Before irreversibly stopping fluorescence, a single LH2 complex in phospholipid bilayer of dimyristoylphosphatidylcholine (DMPC) emitted on average 4 times more fluorescence photons than a complex in detergent micelle of octylglucopyranoside (OG). Fluorescence-excitation spectrum of single LH2 complexes taken at 5 K showed that the LH2 complex is structurally less perturbed in DMPC bilayer than in OG micelle, suggesting that structural instability reduces photoresistance of LH2.

  8. Heat capacity changes in carbohydrates and protein-carbohydrate complexes.

    PubMed

    Chavelas, Eneas A; García-Hernández, Enrique

    2009-05-13

    Carbohydrates are crucial for living cells, playing myriads of functional roles that range from being structural or energy-storage devices to molecular labels that, through non-covalent interaction with proteins, impart exquisite selectivity in processes such as molecular trafficking and cellular recognition. The molecular bases that govern the recognition between carbohydrates and proteins have not been fully understood yet. In the present study, we have obtained a surface-area-based model for the formation heat capacity of protein-carbohydrate complexes, which includes separate terms for the contributions of the two molecular types. The carbohydrate model, which was calibrated using carbohydrate dissolution data, indicates that the heat capacity contribution of a given group surface depends on its position in the saccharide molecule, a picture that is consistent with previous experimental and theoretical studies showing that the high abundance of hydroxy groups in carbohydrates yields particular solvation properties. This model was used to estimate the carbohydrate's contribution in the formation of a protein-carbohydrate complex, which in turn was used to obtain the heat capacity change associated with the protein's binding site. The model is able to account for protein-carbohydrate complexes that cannot be explained using a previous model that only considered the overall contribution of polar and apolar groups, while allowing a more detailed dissection of the elementary contributions that give rise to the formation heat capacity effects of these adducts.

  9. Trichomonas vaginalis: the adhesins AP51 and AP65 bind heme and hemoglobin.

    PubMed

    Ardalan, Shahed; Lee, B Craig; Garber, Gary E

    2009-04-01

    Trichomonas vaginalis is the cause of human trichomoniasis, the most common non-viral sexually transmitted disease worldwide. Although acquisition of iron by binding to host hemoglobin through distinct receptor(s) has been described, no specific heme- or hemoglobin-binding site has been reported in this parasite. To determine the presence of hemoglobin-binding protein(s), membrane proteins were subjected to hemoglobin-affinity chromatography. Eluted proteins were analysed by SDS-PAGE. Two protein bands of 48 and 63 kDa were detected. Competition assay with an excess amount of hemoglobin or hemin in hemoglobin-affinity chromatography could block the 63- and 48-kDa bands, respectively. Further analysis by mass spectrometry indicated that the 48- and 63-kDa proteins had identity with two T. vaginalis adhesins: AP51 and AP65, respectively. This study confirms the existence of multifunctional proteins in T. vaginalis, and suggested that AP51 and AP65, besides serving as adhesion molecules, could also act as heme- and hemoglobin-binding proteins.

  10. Electrophoretic separation of proteins via complexation with a polyelectrolyte

    NASA Astrophysics Data System (ADS)

    Baskin, E. M.; Shklovskii, B. I.; Zilberstein, G. V.

    2003-01-01

    We suggest to augment standard isoelectric focusing for separation of proteins in a gradient of pH by a similar focusing in the presence of a strongly charged polyelectrolyte (PE). Proteins which have the same isoelectric point but different “hidden” charges of both signs in this point make complexes with PE, which focus in different pH. This is a result of charge inversion of such proteins by adsorbed PE molecules, which is sensitive to the hidden charge. Hence, the hidden charge is a new separation parameter.

  11. Atomic force and super-resolution microscopy support a role for LapA as a cell-surface biofilm adhesin of Pseudomonas fluorescens

    PubMed Central

    Ivanov, Ivan E.; Boyd, Chelsea D.; Newell, Peter D.; Schwartz, Mary E.; Turnbull, Lynne; Johnson, Michael S.; Whitchurch, Cynthia B.; O’Toole, George A.; Camesano, Terri A.

    2012-01-01

    Pseudomonas fluorescence Pf0-1 requires the large repeat protein LapA for stable surface attachment. This study presents direct evidence that LapA is a cell-surface-localized adhesin. Atomic force microscopy (AFM) revealed a significant twofold reduction in adhesion force for mutants lacking the LapA protein on the cell surface compared to the wild-type strain. Deletion of lapG, a gene encoding a periplasmic cysteine protease that functions to release LapA from the cell surface, resulted in a twofold increase in the force of adhesion. Three-dimensional structured illumination microscopy (3D-SIM) revealed the presence of the LapA protein on the cell surface, consistent with its role as an adhesin. The protein is only visualized in the cytoplasm for a mutant of the ABC transporter responsible for translocating LapA to the cell surface. Together, these data highlight the power of combining the use of AFM and 3D-SIM with genetic studies to demonstrate that LapA, a member of a large group of RTX-like repeat proteins, is a cell-surface adhesin. PMID:23064158

  12. Biorecognition of Escherichia coli K88 adhesin for glycated porcine albumin.

    PubMed

    Sarabia-Sainz, Andre-i; Ramos-Clamont, Gabriela; Candia-Plata, Ma María del Carmen; Vázquez-Moreno, Luz

    2009-03-01

    Escherichia coli (E. coli) that expresses galactose-reactive lectins, like K88 adhesin, causes high mortality among piglets. Carbohydrates that compete for adhesion could serve as an alternative for disease prevention. Porcine serum albumin (PSA) was modified by non-enzymatic glycation with lactose to produce PSA-Lac or PSA-Glc beta (1-4) Gal, as confirmed by reduction of available free amino groups, increased molecular mass and by Ricinus communis lectin recognition. E. coli K88 binds to PSA-Lac treatments containing three and four lactoses, respectively. In addition, PSA-Lac partially inhibited K88 strain adherence to mucins. These results suggest that neoglycoconjugates obtained by non-enzymatic glycation of proteins may serve in the prophylaxis of piglets' diarrhea.

  13. Characterization of plasmid-borne afa-3 gene clusters encoding afimbrial adhesins expressed by Escherichia coli strains associated with intestinal or urinary tract infections.

    PubMed

    Le Bouguenec, C; Garcia, M I; Ouin, V; Desperrier, J M; Gounon, P; Labigne, A

    1993-12-01

    The afa gene clusters encode afimbrial adhesins (AFA) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains and belong to a family of hemagglutinins recognizing the Dr blood group antigen as a receptor. This family so far includes AFA-I and AFA-III as well as the Dr and F1845 adhesins (B. Nowicki, A. Labigne, S. Moseley, R. Hull, S. Hull, and J. Moulds, Infect. Immun. 58:279-281, 1990). Reported in this work is the genetic organization of the afa-3 gene cluster cloned from a uropathogenic E. coli strain (A30) which expressed a subtype of AFA designated AFA-III. The amino acid sequence of AFA-III was deduced from the nucleotide sequence of the afaE3 gene and was found to be highly homologous to that of the Dr adhesin (98.1% identity). A polymerase chain reaction assay was developed to detect the presence of afa-3 gene clusters in E. coli strains. Study of the genetic support of the afa-3 gene clusters in the strains which showed positive amplification revealed that they were always located on large, 100-kb plasmids whether the strains originated from patients with cystitis or with diarrhea. Moreover, the cloned afa-3 gene clusters from A30 and from the diarrhea-associated strain AL845 appeared to be carried by 9-kb plasmid regions which displayed a similar genetic organization. Chloramphenicol was reported to be a potent inhibitor of receptor binding by the Dr adhesin (Nowicki et al., Infect. Immun. 58:279-281, 1990). AFA-III expressed by strains AL845 and AL847 appeared to mediate, like the Dr adhesin, chloramphenicol-sensitive hemagglutination, whereas AFA-III produced by A30 conferred chloramphenicol-resistant adherence. A comparison of the sequences of these four proteins indicated that the amino acid at position 52 of the processed AFA could be part of the receptor-binding domain.

  14. Unwrapping of DNA-protein complexes under external stretching.

    PubMed

    Sakaue, Takahiro; Löwen, Hartmut

    2004-08-01

    A DNA-protein complex modeled by a semiflexible chain and an attractive spherical core is studied in the situation when an external stretching force is acting on one end monomer of the chain while the other end monomer is kept fixed in space. Without a stretching force, the chain is wrapped around the core. By applying an external stretching force, unwrapping of the complex is induced. We study the statics and dynamics of the unwrapping process by computer simulations and simple phenomenological theory. We find two different scenarios depending on the chain stiffness: For a flexible chain, the extension of the complex scales linearly with the external force applied. The sphere-chain complex is disordered; i.e., there is no clear winding of the chain around the sphere. For a stiff chain, on the other hand, the complex structure is ordered, which is reminiscent of nucleosome. There is a clear winding number, and the unwrapping process under external stretching is discontinuous with jumps of the distance-force curve. This is associated with discrete unwinding processes of the complex. Our predictions are of relevance for experiments, which measure force-extension curves of DNA-protein complexes, such as nucleosome, using optical tweezers.

  15. Protein corona - from molecular adsorption to physiological complexity.

    PubMed

    Treuel, Lennart; Docter, Dominic; Maskos, Michael; Stauber, Roland H

    2015-01-01

    In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP-protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs.

  16. Protein corona – from molecular adsorption to physiological complexity

    PubMed Central

    Docter, Dominic; Maskos, Michael

    2015-01-01

    Summary In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP–protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs. PMID:25977856

  17. Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

    ERIC Educational Resources Information Center

    Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

    2005-01-01

    In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

  18. Ca2+-stabilized adhesin helps an Antarctic bacterium reach out and bind ice.

    PubMed

    Vance, Tyler D R; Olijve, Luuk L C; Campbell, Robert L; Voets, Ilja K; Davies, Peter L; Guo, Shuaiqi

    2014-07-04

    The large size of a 1.5-MDa ice-binding adhesin [MpAFP (Marinomonas primoryensis antifreeze protein)] from an Antarctic Gram-negative bacterium, M. primoryensis, is mainly due to its highly repetitive RII (Region II). MpAFP_RII contains roughly 120 tandem copies of an identical 104-residue repeat. We have previously determined that a single RII repeat folds as a Ca2+-dependent immunoglobulin-like domain. Here, we solved the crystal structure of RII tetra-tandemer (four tandem RII repeats) to a resolution of 1.8 Å. The RII tetra-tandemer reveals an extended (~190-Å × ~25-Å), rod-like structure with four RII-repeats aligned in series with each other. The inter-repeat regions of the RII tetra-tandemer are strengthened by Ca2+ bound to acidic residues. SAXS (small-angle X-ray scattering) profiles indicate the RII tetra-tandemer is significantly rigidified upon Ca2+ binding, and that the protein's solution structure is in excellent agreement with its crystal structure. We hypothesize that >600 Ca2+ help rigidify the chain of ~120 104-residue repeats to form a ~0.6 μm rod-like structure in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed extender role of RII can help the strictly aerobic, motile bacterium bind ice in the upper reaches of the Antarctic lake where oxygen and nutrients are most abundant. Ca2+-induced rigidity of tandem Ig-like repeats in large adhesins might be a general mechanism used by bacteria to bind to their substrates and help colonize specific niches.

  19. Ca2+-stabilized adhesin helps an Antarctic bacterium reach out and bind ice

    PubMed Central

    Vance, Tyler D. R.; Olijve, Luuk L. C.; Campbell, Robert L.; Voets, Ilja K.; Davies, Peter L.; Guo, Shuaiqi

    2014-01-01

    The large size of a 1.5-MDa ice-binding adhesin [MpAFP (Marinomonas primoryensis antifreeze protein)] from an Antarctic Gram-negative bacterium, M. primoryensis, is mainly due to its highly repetitive RII (Region II). MpAFP_RII contains roughly 120 tandem copies of an identical 104-residue repeat. We have previously determined that a single RII repeat folds as a Ca2+-dependent immunoglobulin-like domain. Here, we solved the crystal structure of RII tetra-tandemer (four tandem RII repeats) to a resolution of 1.8 Å. The RII tetra-tandemer reveals an extended (~190-Å × ~25-Å), rod-like structure with four RII-repeats aligned in series with each other. The inter-repeat regions of the RII tetra-tandemer are strengthened by Ca2+ bound to acidic residues. SAXS (small-angle X-ray scattering) profiles indicate the RII tetra-tandemer is significantly rigidified upon Ca2+ binding, and that the protein's solution structure is in excellent agreement with its crystal structure. We hypothesize that >600 Ca2+ help rigidify the chain of ~120 104-residue repeats to form a ~0.6 μm rod-like structure in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed extender role of RII can help the strictly aerobic, motile bacterium bind ice in the upper reaches of the Antarctic lake where oxygen and nutrients are most abundant. Ca2+-induced rigidity of tandem Ig-like repeats in large adhesins might be a general mechanism used by bacteria to bind to their substrates and help colonize specific niches. PMID:24892750

  20. Identification of the mucin-binding adhesin of Pseudomonas cepacia isolated from patients with cystic fibrosis.

    PubMed Central

    Sajjan, S U; Forstner, J F

    1992-01-01

    In previous experiments, we have shown that isolates of Pseudomonas cepacia from sputa of patients with cystic fibrosis (CF), particularly those with severe lung infection, exhibited specific binding to purified respiratory or intestinal mucins (U. Sajjan, M. Corey, M. Karmali, and J. Forstner, J. Clin. Invest. 89:648-656, 1992). The present report describes the identification of the adhesin as a protein located on fimbriae of mucin-binding P. cepacia. From a total of 53 isolates available (from 22 patients with CF), we used three mucin-binding and three non-mucin-binding isolates for our experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude P. cepacia homogenates was performed, the separated proteins were blotted onto nitrocellulose and overlaid with purified mucin, and mucin-binding components were detected with an antimucin antibody and then a second-antibody-alkaline phosphatase conjugate system. Only mucin-binding isolates exhibited a positively stained band at an Mr of 22,000. The 22-kDa protein was purified, and a polyclonal antibody specific for it was developed in rabbits. By electron microscopy and immunogold labelling, both the antibody and mucin (separately) were localized to pili present over the entire surface of the bacterial cells. Non-mucin-binding isolates did not have (or had very few) pili and did not stain with either mucin or the antibody to the 22-kDa protein. The purified 22-kDa protein and its antibody were each able to inhibit piliated P. cepacia binding to mucin. The amino acid composition of the 22-kDa protein was dissimilar to those of the major pilin proteins of Escherichia coli (type 1 pilus) and P. aeruginosa (PAK and PAO1 strains). Both the pili of P. aeruginosa PAK and PAO1 and antibodies to these pili failed to inhibit P. cepacia binding to mucin. Thus, P. cepacia adhesion to mucin is mediated by a pilin-associated 22-kDa protein which differs from epithelial-cell-binding pilin proteins of P. aeruginosa

  1. A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

    PubMed Central

    Lockard, Meghan A.; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2011-01-01

    We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit. PMID:21642284

  2. A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.

    PubMed

    Lockard, Meghan A; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B; Terwilliger, Thomas C; Waldo, Geoffrey S

    2011-07-01

    We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1-10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon(®) resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length 'breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit.

  3. Comparative Study of Elastic Network Model and Protein Contact Network for Protein Complexes: The Hemoglobin Case

    PubMed Central

    Di Paola, Luisa; Liang, Zhongjie; Giuliani, Alessandro

    2017-01-01

    The overall topology and interfacial interactions play key roles in understanding structural and functional principles of protein complexes. Elastic Network Model (ENM) and Protein Contact Network (PCN) are two widely used methods for high throughput investigation of structures and interactions within protein complexes. In this work, the comparative analysis of ENM and PCN relative to hemoglobin (Hb) was taken as case study. We examine four types of structural and dynamical paradigms, namely, conformational change between different states of Hbs, modular analysis, allosteric mechanisms studies, and interface characterization of an Hb. The comparative study shows that ENM has an advantage in studying dynamical properties and protein-protein interfaces, while PCN is better for describing protein structures quantitatively both from local and from global levels. We suggest that the integration of ENM and PCN would give a potential but powerful tool in structural systems biology. PMID:28243596

  4. Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry.

    PubMed

    Li, Huilin; Sheng, Yuewei; McGee, William; Cammarata, Michael; Holden, Dustin; Loo, Joseph A

    2017-03-07

    Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

  5. Transmembrane protein 147 (TMEM147) is a novel component of the Nicalin-NOMO protein complex.

    PubMed

    Dettmer, Ulf; Kuhn, Peer-Hendrik; Abou-Ajram, Claudia; Lichtenthaler, Stefan F; Krüger, Marcus; Kremmer, Elisabeth; Haass, Christian; Haffner, Christof

    2010-08-20

    Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely gamma-secretase and the Nicalin-NOMO (Nodal modulator) complex. The gamma-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the beta-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200-220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A approximately 22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to gamma-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with gamma-secretase.

  6. Transmembrane Protein 147 (TMEM147) Is a Novel Component of the Nicalin-NOMO Protein Complex*

    PubMed Central

    Dettmer, Ulf; Kuhn, Peer-Hendrik; Abou-Ajram, Claudia; Lichtenthaler, Stefan F.; Krüger, Marcus; Kremmer, Elisabeth; Haass, Christian; Haffner, Christof

    2010-01-01

    Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely γ-secretase and the Nicalin-NOMO (Nodal modulator) complex. The γ-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the β-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200–220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A ∼22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to γ-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with γ-secretase. PMID:20538592

  7. Modeling of Protein Binary Complexes Using Structural Mass Spectrometry Data

    SciTech Connect

    Amisha Kamal,J.; Chance, M.

    2008-01-01

    In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints--positive and/or negative--in the docking step and are also used to decide the type of energy filter--electrostatics or desolvation--in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure.

  8. Exploration of the Dynamic Properties of Protein Complexes Predicted from Spatially Constrained Protein-Protein Interaction Networks

    PubMed Central

    Yen, Eric A.; Tsay, Aaron; Waldispuhl, Jerome; Vogel, Jackie

    2014-01-01

    Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and visualization of the hidden

  9. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*

    PubMed Central

    Turk, Rolf; Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Pospisil, Tyler C.; Jones, Kayla S.; Campbell, Kevin P.; Wright, Michael E.

    2016-01-01

    Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research. PMID:27099343

  10. Heterodimeric Drosophila gap gene protein complexes acting as transcriptional repressors.

    PubMed Central

    Sauer, F; Jäckle, H

    1995-01-01

    The Drosophila gap gene Krüppel (Kr) encodes a transcriptional regulator. It acts both as an integral part of the Drosophila segmentation gene in the early blastoderm and in a variety of tissues and organs at later stages of embryogenesis. In transfected tissue culture cells, the Kr protein (Kr) was shown to both activate and repress gene expression in a concentration-dependent manner when acting from a single binding site close to the promoter. Here we show that KR can associate with the transcription factors encoded by the gap genes knirps (kni) and hunchback (hb) which affect KR-dependent gene expression in Drosophila tissue culture cells. The association of DNA-bound hb protein or free kni protein with distinct but different regions of KR results in the formation of DNA-bound transcriptional repressor complexes. Our results suggest that individual transcription factors can associate to form protein complexes which act as direct repressors of transcription. The interactions shown here add an unexpected level of complexity to the control of gene expression. Images PMID:7588607

  11. Characterization of the human GARP (Golgi associated retrograde protein) complex

    SciTech Connect

    Liewen, Heike; Meinhold-Heerlein, Ivo; Oliveira, Vasco; Schwarzenbacher, Robert; Luo Guorong; Wadle, Andreas; Jung, Martin; Pfreundschuh, Michael; Stenner-Liewen, Frank . E-mail: stenlie@t-online.de

    2005-05-15

    The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.

  12. Changes in protein structure at the interface accompanying complex formation.

    PubMed

    Chakravarty, Devlina; Janin, Joël; Robert, Charles H; Chakrabarti, Pinak

    2015-11-01

    Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U) and bound (B) forms of protein structures from the Protein-Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA), secondary structure, temperature factors (B factors) and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69%) of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered) residues in the U form which are observed (ordered) in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial.

  13. Comparison of Surface Proteomes of Adherence Variants of Listeria Monocytogenes Using LC-MS/MS for Identification of Potential Surface Adhesins

    PubMed Central

    Tiong, Hung King; Hartson, Steven D.; Muriana, Peter M.

    2016-01-01

    The ability of Listeria monocytogenes to adhere and form biofilms leads to persistence in food processing plants and food-associated listeriosis. The role of specific surface proteins as adhesins to attach Listeria cells to various contact surfaces has not been well characterized to date. In prior research comparing different methods for surface protein extraction, the Ghost urea method revealed cleaner protein content as verified by the least cytoplasmic protein detected in surface extracts using LC-MS/MS. The same technique was utilized to extract and detect surface proteins among two surface-adherent phenotypic strains of L. monocytogenes (i.e., strongly and weakly adherent). Of 640 total proteins detected among planktonic and sessile cells, 21 protein members were exclusively detected in the sessile cells. Relative LC-MS/MS detection and quantification of surface-extracted proteins from the planktonic weakly adherent (CW35) and strongly adherent strains (99-38) were examined by protein mass normalization of proteins. We found that L. monocytogenes 99-38 exhibited a total of 22 surface proteins that were over-expressed: 11 proteins were detected in surface extracts of both sessile and planktonic 99-38 that were ≥5-fold over-expressed while another 11 proteins were detected only in planktonic 99-38 cells that were ≥10-fold over-expressed. Our results suggest that these protein members are worthy of further investigation for their involvement as surface adhesins. PMID:27196934

  14. The RCP-Rab11 complex regulates endocytic protein sorting.

    PubMed

    Peden, Andrew A; Schonteich, Eric; Chun, John; Junutula, Jagath R; Scheller, Richard H; Prekeris, Rytis

    2004-08-01

    Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP-Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes.

  15. 2D depiction of nonbonding interactions for protein complexes.

    PubMed

    Zhou, Peng; Tian, Feifei; Shang, Zhicai

    2009-04-30

    A program called the 2D-GraLab is described for automatically generating schematic representation of nonbonding interactions across the protein binding interfaces. The input file of this program takes the standard PDB format, and the outputs are two-dimensional PostScript diagrams giving intuitive and informative description of the protein-protein interactions and their energetics properties, including hydrogen bond, salt bridge, van der Waals interaction, hydrophobic contact, pi-pi stacking, disulfide bond, desolvation effect, and loss of conformational entropy. To ensure these interaction information are determined accurately and reliably, methods and standalone programs employed in the 2D-GraLab are all widely used in the chemistry and biology community. The generated diagrams allow intuitive visualization of the interaction mode and binding specificity between two subunits in protein complexes, and by providing information on nonbonding energetics and geometric characteristics, the program offers the possibility of comparing different protein binding profiles in a detailed, objective, and quantitative manner. We expect that this 2D molecular graphics tool could be useful for the experimentalists and theoreticians interested in protein structure and protein engineering.

  16. Homology modelling of protein-protein complexes: a simple method and its possibilities and limitations

    PubMed Central

    Launay, Guillaume; Simonson, Thomas

    2008-01-01

    Background Structure-based computational methods are needed to help identify and characterize protein-protein complexes and their function. For individual proteins, the most successful technique is homology modelling. We investigate a simple extension of this technique to protein-protein complexes. We consider a large set of complexes of known structures, involving pairs of single-domain proteins. The complexes are compared with each other to establish their sequence and structural similarities and the relation between the two. Compared to earlier studies, a simpler dataset, a simpler structural alignment procedure, and an additional energy criterion are used. Next, we compare the Xray structures to models obtained by threading the native sequence onto other, homologous complexes. An elementary requirement for a successful energy function is to rank the native structure above any threaded structure. We use the DFIREβ energy function, whose quality and complexity are typical of the models used today. Finally, we compare near-native models to distinctly non-native models. Results If weakly stable complexes are excluded (defined by a binding energy cutoff), as well as a few unusual complexes, a simple homology principle holds: complexes that share more than 35% sequence identity share similar structures and interaction modes; this principle was less clearcut in earlier studies. The energy function was then tested for its ability to identify experimental structures among sets of decoys, produced by a simple threading procedure. On average, the experimental structure is ranked above 92% of the alternate structures. Thus, discrimination of the native structure is good but not perfect. The discrimination of near-native structures is fair. Typically, a single, alternate, non-native binding mode exists that has a native-like energy. Some of the associated failures may correspond to genuine, alternate binding modes and/or native complexes that are artefacts of the crystal

  17. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  18. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    NASA Astrophysics Data System (ADS)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  19. A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.

    PubMed Central

    Yeakley, J M; Morfin, J P; Rosenfeld, M G; Fu, X D

    1996-01-01

    A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8755518

  20. The role of filamentous hemagglutinin adhesin in adherence and biofilm formation in Acinetobacter baumannii ATCC19606(T).

    PubMed

    Darvish Alipour Astaneh, Shakiba; Rasooli, Iraj; Mousavi Gargari, Seyed Latif

    2014-09-01

    Filamentous hemagglutinin adhesins (FHA) are key factors for bacterial attachment and subsequent cell accumulation on substrates. Here an FHA-like Outer membrane (OM) adhesin of Acinetobacter baumannii ATCC19606(T) was displayed on Escherichia coli. The candidate autotransporter (AT) genes were identified in A. baumannii ATCC19606(T) genome. The exoprotein (FhaB1) and transporter (FhaC1) were produced independently within the same cell (FhaB1C1). The fhaC1 was mutated. In vitro adherence to epithelial cells of the recombinant FhaB1C1 and the mutant strains were compared with A. baumanni ATCC19606(T). A bivalent chimeric protein (K) composed of immunologically important portions of fhaB1 (B) and fhaC1 (C) was constructed. The mice vaccinated with chimeric protein were challenged with A. baumannii ATCC19606(T) and FhaB1C1 producing recombinant E. coli. Mutations in the fhaC1 resulted in the absence of FhaB1 in the OM. Expression of FhaB1C1 enhanced the adherence of recombinant bacteria to A546 bronchial cell line. The results revealed association of FhaB1 with bacterial adhesion and biofilm formation. Immunization with a combination of recombinant B and K proteins proved protective against A. baumanni ATCC19606(T). The findings may be applied in active and passive immunization strategies against A. baumannii.

  1. Changes in protein structure at the interface accompanying complex formation

    PubMed Central

    Chakravarty, Devlina; Janin, Joël; Robert, Charles H.; Chakrabarti, Pinak

    2015-01-01

    Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U) and bound (B) forms of protein structures from the Protein–Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA), secondary structure, temperature factors (B factors) and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69%) of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered) residues in the U form which are observed (ordered) in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial. PMID:26594372

  2. Protein Complex Affinity Capture from Cryomilled Mammalian Cells.

    PubMed

    LaCava, John; Jiang, Hua; Rout, Michael P

    2016-12-09

    Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

  3. Protein Complex Affinity Capture from Cryomilled Mammalian Cells

    PubMed Central

    LaCava, John; Jiang, Hua; Rout, Michael P.

    2016-01-01

    Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets. PMID:28060343

  4. Membrane protein architects: the role of the BAM complex in outer membrane protein assembly.

    PubMed

    Knowles, Timothy J; Scott-Tucker, Anthony; Overduin, Michael; Henderson, Ian R

    2009-03-01

    The folding of transmembrane proteins into the outer membrane presents formidable challenges to Gram-negative bacteria. These proteins must migrate from the cytoplasm, through the inner membrane and into the periplasm, before being recognized by the beta-barrel assembly machinery, which mediates efficient insertion of folded beta-barrels into the outer membrane. Recent discoveries of component structures and accessory interactions of this complex are yielding insights into how cells fold membrane proteins. Here, we discuss how these structures illuminate the mechanisms responsible for the biogenesis of outer membrane proteins.

  5. The Streptococcus pneumoniae adhesin PsrP binds to Keratin 10 on lung cells

    PubMed Central

    Shivshankar, Pooja; Sanchez, Carlos; Rose, Lloyd F.; Orihuela, Carlos J.

    2009-01-01

    Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island encoded adhesin that mediates attachment to lung cells. It is a member of the Serine-rich repeat protein (SRRP) family and the largest bacterial protein known. PsrP production by S. pneumoniae was confirmed by immunoblotting and a truncated version of the protein was determined to be glycosylated. Using isogenic psrP mutants complemented with various PsrP constructs and competitive inhibition assays with recombinant proteins, we determined that PsrP requires an extended SRR2 domain for function and that adhesion is mediated through amino acids 273-341 of its Basic Region (BR) domain. Affinity chromatography, immunoprecipitation, ELISA, FACS, and immunofluorescent co-localization studies determined that PsrP binds to Keratin 10 (K10) on the surface of lung but not nasopharyngeal epithelial cells. Unglycosylated K10 bound to wild type but not psrP deficient pneumococci; suggesting that unlike other SRRPs, PsrP-mediated adhesion was independent of lectin activity. Finally, mice immunized with recombinant (r)PsrPBR had significantly less bacteria in their blood and improved survival versus controls following intranasal challenge. We conclude that the BR domain of PsrP binds to K10 in a lectin-independent manner; that K10 is expressed on lung cells; and that vaccination with rPsrPBR is protective against pneumococcal disease. PMID:19627498

  6. Supercharging Protein Complexes from Aqueous Solution Disrupts their Native Conformations

    NASA Astrophysics Data System (ADS)

    Sterling, Harry J.; Kintzer, Alexander F.; Feld, Geoffrey K.; Cassou, Catherine A.; Krantz, Bryan A.; Williams, Evan R.

    2012-02-01

    The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol ( m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared with the "native" complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a prechannel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of Coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets, where enrichment of the supercharging reagent during droplet evaporation occurs.

  7. A profile of protein-protein interaction: Crystal structure of a lectin-lectin complex.

    PubMed

    Surya, Sukumaran; Abhilash, Joseph; Geethanandan, Krishnan; Sadasivan, Chittalakkottu; Haridas, Madhathilkovilakathu

    2016-06-01

    Proteins may utilize complex networks of interactions to create/proceed signaling pathways of highly adaptive responses such as programmed cell death. Direct binary interactions study of proteins may help propose models for protein-protein interaction. Towards this goal we applied a combination of thermodynamic kinetics and crystal structure analyses to elucidate the complexity and diversity in such interactions. By determining the heat change on the association of two galactose-specific legume lectins from Butea monosperma (BML) and Spatholobus parviflorus (SPL) belonging to Fabaceae family helped to compute the binding equilibrium. It was extended further by X-ray structural analysis of BML-SPL binary complex. In order to chart the proteins interacting mainly through their interfaces, identification of the nature of forces which stabilized the association of the lectin-lectin complex was examined. Comprehensive analysis of the BMLSPL complex by isothermal titration calorimetry and X-ray crystal structure threw new light on the lectin-lectin interactions suggesting of their use in diverse areas of glycobiology.

  8. Characterization of known protein complexes using k-connectivity and other topological measures

    PubMed Central

    Gallagher, Suzanne R; Goldberg, Debra S

    2015-01-01

    Many protein complexes are densely packed, so proteins within complexes often interact with several other proteins in the complex. Steric constraints prevent most proteins from simultaneously binding more than a handful of other proteins, regardless of the number of proteins in the complex. Because of this, as complex size increases, several measures of the complex decrease within protein-protein interaction networks. However, k-connectivity, the number of vertices or edges that need to be removed in order to disconnect a graph, may be consistently high for protein complexes. The property of k-connectivity has been little used previously in the investigation of protein-protein interactions. To understand the discriminative power of k-connectivity and other topological measures for identifying unknown protein complexes, we characterized these properties in known Saccharomyces cerevisiae protein complexes in networks generated both from highly accurate X-ray crystallography experiments which give an accurate model of each complex, and also as the complexes appear in high-throughput yeast 2-hybrid studies in which new complexes may be discovered. We also computed these properties for appropriate random subgraphs.We found that clustering coefficient, mutual clustering coefficient, and k-connectivity are better indicators of known protein complexes than edge density, degree, or betweenness. This suggests new directions for future protein complex-finding algorithms. PMID:26913183

  9. DMS Footprinting of Structured RNAs and RNA-Protein Complexes

    PubMed Central

    Tijerina, Pilar; Mohr, Sabine; Russell, Rick

    2008-01-01

    We describe a protocol in which dimethyl sulfate (DMS) modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA-protein complexes (RNPs). The protocol is optimized for RNAs of small to moderate size (≤500 nucleotides). The RNA or RNP is first exposed to DMS under conditions that promote formation of the folded structure or complex, as well as ‘control’ conditions that do not allow folding or complex formation. The positions and extents of modification are then determined by primer extension, polyacrylamide gel electrophoresis (PAGE), and quantitative analysis. From changes in the extent of modification upon folding or protein binding (appearance of a ‘footprint’), it is possible to detect local changes in RNA secondary and tertiary structure, as well as the formation of RNA-protein contacts. This protocol takes 1.5–3 days to complete, depending on the type of analysis used. PMID:17948004

  10. Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations

    PubMed Central

    Waadt, Rainer; Schlücking, Kathrin; Schroeder, Julian I.; Kudla, Jörg

    2014-01-01

    Summary The analyses of protein-protein interactions is crucial for understanding cellular processes including signal transduction, protein trafficking and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms, and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further we discuss different BiFC applications and provide examples for proper BiFC analyses in planta. PMID:24057390

  11. Radioprotection by polyethylene glycol-protein complexes in mice

    SciTech Connect

    Gray, B.H.; Stull, R.W.

    1983-03-01

    Polyethylene glycol of about 5000 D was activated with cyanuric chloride, and the activated compound was complexed to each of three proteins. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase were each radioprotectants when administered prophylactically to female B6CBF1 mice before irradiation. The dose reduction factor for these mice was 1.2 when 5000 units of polyethylene glycol-catalase was administered before /sup 60/Co irradiation. Female B6CBF1 mice administered prophylactic intravenous injections of catalase, polyethylene glycol-albumin, or heat-denatured polyethylene glycol-catalase had survival rates similar to phosphate-buffered saline-injected control mice following /sup 60/Co irradiation. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase have radioprotective activity in B6CBF1 mice, which appears to depend in part on enzymatic activities of the complex. However, no radioprotective effect was observed in male C57BL/6 mice injected with each polyethylene glycol-protein complex at either 3 or 24 hr before irradiation. The mechanism for radioprotection by these complexes may depend in part on other factors.

  12. Small-Angle X-Ray Scattering From RNA, Proteins, And Protein Complexes

    SciTech Connect

    Lipfert, Jan; Doniach, Sebastian; /Stanford U., Phys. Dept. /Stanford U., Appl. Phys. Dept. /SLAC, SSRL

    2007-09-18

    Small-angle X-ray scattering (SAXS) is increasingly used to characterize the structure and interactions of biological macromolecules and their complexes in solution. Although still a low-resolution technique, the advent of high-flux synchrotron sources and the development of algorithms for the reconstruction of 3-D electron density maps from 1-D scattering profiles have made possible the generation of useful low-resolution molecular models from SAXS data. Furthermore, SAXS is well suited for the study of unfolded or partially folded conformational ensembles as a function of time or solution conditions. Here, we review recently developed algorithms for 3-D structure modeling and applications to protein complexes. Furthermore, we discuss the emerging use of SAXS as a tool to study membrane protein-detergent complexes. SAXS is proving useful to study the folding of functional RNA molecules, and finally we discuss uses of SAXS to study ensembles of denatured proteins.

  13. Integrating computational methods and experimental data for understanding the recognition mechanism and binding affinity of protein-protein complexes.

    PubMed

    Gromiha, M Michael; Yugandhar, K

    2017-01-07

    Protein-protein interactions perform several functions inside the cell. Understanding the recognition mechanism and binding affinity of protein-protein complexes is a challenging problem in experimental and computational biology. In this review, we focus on two aspects (i) understanding the recognition mechanism and (ii) predicting the binding affinity. The first part deals with computational techniques for identifying the binding site residues and the contribution of important interactions for understanding the recognition mechanism of protein-protein complexes in comparison with experimental observations. The second part is devoted to the methods developed for discriminating high and low affinity complexes, and predicting the binding affinity of protein-protein complexes using three-dimensional structural information and just from the amino acid sequence. The overall view enhances our understanding of the integration of experimental data and computational methods, recognition mechanism of protein-protein complexes and the binding affinity.

  14. Cooperative role for tetraspanins in adhesin-mediated attachment of bacterial species to human epithelial cells.

    PubMed

    Green, Luke R; Monk, Peter N; Partridge, Lynda J; Morris, Paul; Gorringe, Andrew R; Read, Robert C

    2011-06-01

    The tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins. Neisseria meningitidis, the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function in Neisseria meningitidis adherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence of Neisseria meningitidis. In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells of Neisseria meningitidis strain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains of Staphylococcus aureus, Neisseria lactamica, Escherichia coli, and Streptococcus pneumoniae was also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors for Neisseria meningitidis and potentially for multiple species of bacteria.

  15. BslA, the S-layer adhesin of B. anthracis, is a virulence factor for anthrax pathogenesis.

    PubMed

    Kern, Justin; Schneewind, Olaf

    2010-01-01

    Microbial pathogens use adhesive surface proteins to bind to and interact with host tissues, events that are universal for the pathogenesis of infectious diseases. A surface adhesin of Bacillus anthracis, the causative agent of anthrax, required to mediate these steps has not been discovered. Previous work identified BslA, an S-layer protein, to be necessary and sufficient for adhesion of the anthrax vaccine strain, Bacillus anthracis Sterne, to host cells. Here we asked whether encapsulated bacilli require BslA for anthrax pathogenesis in guinea pigs. Compared with the highly virulent parent strain B. anthracis Ames, bslA mutants displayed a dramatic increase in the lethal dose and in mean time-to-death. Whereas all tissues of animals infected with B. anthracis Ames contained high numbers of bacilli, only few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant. Surface display of BslA occurred at the poles of encapsulated bacilli and enabled the binding of vegetative forms to host cells. Together these results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames.

  16. Optimization and dynamics of protein-protein complexes using B-splines.

    PubMed

    Gillilan, Richard E; Lilien, Ryan H

    2004-10-01

    A moving-grid approach for optimization and dynamics of protein-protein complexes is introduced, which utilizes cubic B-spline interpolation for rapid energy and force evaluation. The method allows for the efficient use of full electrostatic potentials joined smoothly to multipoles at long distance so that multiprotein simulation is possible. Using a recently published benchmark of 58 protein complexes, we examine the performance and quality of the grid approximation, refining cocrystallized complexes to within 0.68 A RMSD of interface atoms, close to the optimum 0.63 A produced by the underlying MMFF94 force field. We quantify the theoretical statistical advantage of using minimization in a stochastic search in the case of two rigid bodies, and contrast it with the underlying cost of conjugate gradient minimization using B-splines. The volumes of conjugate gradient minimization basins of attraction in cocrystallized systems are generally orders of magnitude larger than well volumes based on energy thresholds needed to discriminate native from nonnative states; nonetheless, computational cost is significant. Molecular dynamics using B-splines is doubly efficient due to the combined advantages of rapid force evaluation and large simulation step sizes. Large basins localized around the native state and other possible binding sites are identifiable during simulations of protein-protein motion. In addition to providing increased modeling detail, B-splines offer new algorithmic possibilities that should be valuable in refining docking candidates and studying global complex behavior.

  17. Biodegradation of the chitin-protein complex in crustacean cuticle

    USGS Publications Warehouse

    Artur, Stankiewicz B.; Mastalerz, Maria; Hof, C.H.J.; Bierstedt, A.; Flannery, M.B.; Briggs, D.E.G.; Evershed, R.P.

    1998-01-01

    Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative analysis of amino acids (by HPLC) and chitin showed that the major loss of proteins and chitin occurred between weeks 1 and 2. After 8 weeks tyrosine, tryptophan and valine are the most prominent amino acid moieties, showing their resistance to degradation. The presence of cyclic ketones in the pyrolysates indicates that mucopolysaccharides or other bound non-chitinous carbohydrates are also resistant to decay. There is no evidence of structural degradation of chitin prior to 8 weeks when FTIR revealed a reduction in chitin-specific bands. The chemical changes are paralleled by structural changes in the cuticle, which becomes an increasingly open structure consisting of loose chitinous fibres. The rapid rate of decay in the experiments suggests that where chitin and protein are preserved in fossil cuticles degradation must have been inhibited.Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative

  18. Protein Complexes are Central in the Yeast Genetic Landscape

    PubMed Central

    Michaut, Magali; Baryshnikova, Anastasia; Costanzo, Michael; Myers, Chad L.; Andrews, Brenda J.; Boone, Charles; Bader, Gary D.

    2011-01-01

    If perturbing two genes together has a stronger or weaker effect than expected, they are said to genetically interact. Genetic interactions are important because they help map gene function, and functionally related genes have similar genetic interaction patterns. Mapping quantitative (positive and negative) genetic interactions on a global scale has recently become possible. This data clearly shows groups of genes connected by predominantly positive or negative interactions, termed monochromatic groups. These groups often correspond to functional modules, like biological processes or complexes, or connections between modules. However it is not yet known how these patterns globally relate to known functional modules. Here we systematically study the monochromatic nature of known biological processes using the largest quantitative genetic interaction data set available, which includes fitness measurements for ∼5.4 million gene pairs in the yeast Saccharomyces cerevisiae. We find that only 10% of biological processes, as defined by Gene Ontology annotations, and less than 1% of inter-process connections are monochromatic. Further, we show that protein complexes are responsible for a surprisingly large fraction of these patterns. This suggests that complexes play a central role in shaping the monochromatic landscape of biological processes. Altogether this work shows that both positive and negative monochromatic patterns are found in known biological processes and in their connections and that protein complexes play an important role in these patterns. The monochromatic processes, complexes and connections we find chart a hierarchical and modular map of sensitive and redundant biological systems in the yeast cell that will be useful for gene function prediction and comparison across phenotypes and organisms. Furthermore the analysis methods we develop are applicable to other species for which genetic interactions will progressively become more available. PMID

  19. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    SciTech Connect

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S. E-mail: mkukrishna@rediffmail.com

    2005-08-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V{sub M}) of 3.3 Å{sup 3} Da{sup −1}, corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

  20. Spectroscopic analysis of protein Fe-NO complexes.

    PubMed

    Bellota-Antón, César; Munnoch, John; Robb, Kirsty; Adamczyk, Katrin; Candelaresi, Marco; Parker, Anthony W; Dixon, Ray; Hutchings, Matthew I; Hunt, Neil T; Tucker, Nicholas P

    2011-10-01

    The toxic free radical NO (nitric oxide) has diverse biological roles in eukaryotes and bacteria, being involved in signalling, vasodilation, blood clotting and immunity, and as an intermediate in microbial denitrification. The predominant biological mechanism of detecting NO is through the formation of iron nitrosyl complexes, although this is a deleterious process for other iron-containing enzymes. We have previously applied techniques such as UV-visible and EPR spectroscopy to the analysis of protein Fe-NO complex formation in order to study how NO controls the activity of the bacterial transcriptional regulators NorR and NsrR. These studies have analysed NO-dependent biological activity both in vitro and in vivo using diverse biochemical, molecular and spectroscopic methods. Recently, we have applied ultrafast 2D-IR (two-dimensional IR) spectroscopy to the analysis of NO-protein interactions using Mb (myoglobin) and Cc (cytochrome c) as model haem proteins. The ultrafast fluctuations of Cc and Mb show marked differences, indicating altered flexibility of the haem pockets. We have extended this analysis to bacterial catalase enzymes that are known to play a role in the nitrosative stress response by detoxifying peroxynitrite. The first 2D-IR analysis of haem nitrosylation and perspectives for the future are discussed.

  1. Inferring drug-disease associations based on known protein complexes.

    PubMed

    Yu, Liang; Huang, Jianbin; Ma, Zhixin; Zhang, Jing; Zou, Yapeng; Gao, Lin

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html.

  2. Inferring drug-disease associations based on known protein complexes

    PubMed Central

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html. PMID:26044949

  3. Adhesins involved in attachment to abiotic surfaces by Gram-negative bacteria

    PubMed Central

    Berne, Cécile; Ducret, Adrien; Hardy, Gail G; Brun, Yves V.

    2015-01-01

    During the first step of biofilm formation, initial attachment is dictated by physicochemical and electrostatic interactions between the surface and the bacterial envelope. Depending upon the nature of these interactions, attachment can be transient or permanent. To achieve irreversible attachment, bacterial cells have developed a series of surface adhesins promoting specific or non-specific adhesion under various environmental conditions. This chapter will review the recent advances in our understanding of the secretion, assembly and regulation of the bacterial adhesins during biofilm formation with a particular emphasis on the fimbrial, non-fimbrial and discrete polysaccharide adhesins in Gram-negative bacteria. PMID:26350310

  4. Anticancer osmium complex inhibitors of the HIF-1α and p300 protein-protein interaction

    PubMed Central

    Yang, Chao; Wang, Wanhe; Li, Guo-Dong; Zhong, Hai-Jing; Dong, Zhen-Zhen; Wong, Chun-Yuen; Kwong, Daniel W. J.; Ma, Dik-Lung; Leung, Chung-Hang

    2017-01-01

    The hypoxia inducible factor (HIF) pathway has been considered to be an attractive anti-cancer target. One strategy to inhibit HIF activity is through the disruption of the HIF-1α–p300 protein-protein interaction. We report herein the identification of an osmium(II) complex as the first metal-based inhibitor of the HIF-1α–p300 interaction. We evaluated the effect of complex 1 on HIF-1α signaling pathway in vitro and in cellulo by using the dual luciferase reporter assay, co-immunoprecipitation assay, and immunoblot assay. Complex 1 exhibited a dose-dependent inhibition of HRE-driven luciferase activity, with an IC50 value of 1.22 μM. Complex 1 interfered with the HIF-1α–p300 interaction as revealed by a dose-dependent reduction of p300 co-precipitated with HIF-1α as the concentration of complex 1 was increased. Complex 1 repressed the phosphorylation of SRC, AKT and STAT3, and had no discernible effect on the activity of NF-κB. We anticipate that complex 1 could be utilized as a promising scaffold for the further development of more potent HIF-1α inhibitors for anti-cancer treatment. PMID:28225008

  5. Analysis of the interface variability in NMR structure ensembles of protein-protein complexes.

    PubMed

    Calvanese, Luisa; D'Auria, Gabriella; Vangone, Anna; Falcigno, Lucia; Oliva, Romina

    2016-06-01

    NMR structures consist in ensembles of conformers, all satisfying the experimental restraints, which exhibit a certain degree of structural variability. We analyzed here the interface in NMR ensembles of protein-protein heterodimeric complexes and found it to span a wide range of different conservations. The different exhibited conservations do not simply correlate with the size of the systems/interfaces, and are most probably the result of an interplay between different factors, including the quality of experimental data and the intrinsic complex flexibility. In any case, this information is not to be missed when NMR structures of protein-protein complexes are analyzed; especially considering that, as we also show here, the first NMR conformer is usually not the one which best reflects the overall interface. To quantify the interface conservation and to analyze it, we used an approach originally conceived for the analysis and ranking of ensembles of docking models, which has now been extended to directly deal with NMR ensembles. We propose this approach, based on the conservation of the inter-residue contacts at the interface, both for the analysis of the interface in whole ensembles of NMR complexes and for the possible selection of a single conformer as the best representative of the overall interface. In order to make the analyses automatic and fast, we made the protocol available as a web tool at: https://www.molnac.unisa.it/BioTools/consrank/consrank-nmr.html.

  6. Force-induced remodelling of proteins and their complexes

    PubMed Central

    Chen, Yun; Radford, Sheena E; Brockwell, David J

    2015-01-01

    Force can drive conformational changes in proteins, as well as modulate their stability and the affinity of their complexes, allowing a mechanical input to be converted into a biochemical output. These properties have been utilised by nature and force is now recognised to be widely used at the cellular level. The effects of force on the biophysical properties of biological systems can be large and varied. As these effects are only apparent in the presence of force, studies on the same proteins using traditional ensemble biophysical methods can yield apparently conflicting results. Where appropriate, therefore, force measurements should be integrated with other experimental approaches to understand the physiological context of the system under study. PMID:25710390

  7. Evidence for a protein-protein complex during iron loading into ferritin by ceruloplasmin.

    PubMed

    Reilly, C A; Sorlie, M; Aust, S D

    1998-06-01

    The formation of a protein-protein complex for the loading of iron into ferritin by ceruloplasmin was investigated. Ferritin stimulated the ferroxidase activity of ceruloplasmin unless the ferritin was fully loaded, in which case it inhibited the ferroxidase activity of ceruloplasmin. The apparent association constant for the interaction of ferritin and ceruloplasmin was 24 nM. Isothermal titration calorimetry indicated that the interaction of ceruloplasmin and ferritin was endothermic, driven by positive changes in entropy. The association constants for complex formation between ferritin and ceruloplasmin were 4.5 +/- 0.7 x 10(5) and 9.5 +/- 0.3 x 10(4) M-1 for the reduced and oxidized forms of ceruloplasmin, respectively. The oxidized form of ceruloplasmin was retained on an affinity column with ferritin immobilized as the ligand and remained bound to the column with mobile phases of increased hydrophobicity, but was eluted with increased ionic strength. The ability of ceruloplasmin to remain bound to the affinity resin was affected by the species from which ceruloplasmin was isolated. Gradient ultracentrifugation also provided evidence that the two proteins were associated, since ferritin promoted migration of ceruloplasmin through the gradient. Including ferrous iron in the gradient resulted in reduction of ceruloplasmin and increased the mobility of ceruloplasmin with ferritin. These data provide evidence that ferritin and ceruloplasmin form a protein-protein complex during iron loading into ferritin, which may limit redox cycling of iron in vivo.

  8. Rediscovery of halogen bonds in protein-ligand complexes.

    PubMed

    Zhou, P; Tian, F; Zou, J; Shang, Z

    2010-04-01

    Although the halogen bond has attracted much interest in chemistry and material science communities, its implications for drug design are just now coming to light. The protein-ligand interactions through short halogen-oxygen/nitrogen/sulfur contacts have been observed in crystal structures for a long time, but only in recent years, with the experimental and theoretical progress in weak biological interactions, especially the pioneering works contributed by Ho and co-workers (Auffinger, P.; Hays, F. A.; Westhof, E.; Ho, P. S. Proc. Natl. Acad. Sci. USA 2004, 101, 16789-16794), these short contacts involving halogens in biomolecules were rediscovered and re-recognized as halogen bonds to stress their shared similarities with hydrogen bonds in strength and directionality. Crystal structure determinations of protein complexes with halogenated ligands preliminarily unveiled the functionality of halogen bonds in protein-ligand recogni-tion. Database surveys further revealed a considerable number of short halogen-oxygen contacts between proteins and halogenated ligands. Theoretical calculations on model and real systems eventually gave a quantitative pronouncement for the substantial contribution of halogen bonds to ligand binding. All of these works forebode that the halogen bond can be exploited as a new and versatile tool for rational drug design and bio-crystal engineering.

  9. The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

    PubMed

    Satoh, Jun-ichi; Onoue, Hiroyuki; Arima, Kunimasa; Yamamura, Takashi

    2005-10-01

    The 14-3-3 protein family consists of acidic 30-kDa proteins composed of 7 isoforms expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 protein identified in the cerebrospinal fluid provides a surrogate marker for premortem diagnosis of Creutzfeldt-Jakob disease, although an active involvement of 14-3-3 in the pathogenesis of prion diseases remains unknown. By protein overlay and mass spectrometric analysis of protein extract of NTera2-derived differentiated neurons, we identified heat shock protein Hsp60 as a 14-3-3-interacting protein. The 14-3-3zeta and gamma isoforms interacted with Hsp60, suggesting that the interaction is not isoform-specific. Furthermore, the interaction was identified in SK-N-SH neuroblastoma, U-373MG astrocytoma, and HeLa cervical carcinoma cells. The cellular prion protein (PrPC) along with Hsp60 was coimmunoprecipitated with 14-3-3 in the human brain protein extract. By protein overlay, 14-3-3 interacted with both recombinant human Hsp60 and PrPC produced by Escherichia coli, indicating that the molecular interaction is phosphorylation-independent. The 14-3-3-binding domain was located in the N-terminal half (NTF) of Hsp60 spanning amino acid residues 27-287 and the NTF of PrPC spanning amino acid residues 23-137. By immunostaining, the 14-3-3 protein Hsp60 and PrPC were colocalized chiefly in the mitochondria of human neuronal progenitor cells in culture, and were coexpressed most prominently in neurons and reactive astrocytes in the human brain. These observations indicate that the 14-3-3 protein forms a molecular complex with Hsp60 and PrPC in the human CNS under physiological conditions and suggest that this complex might become disintegrated in the pathologic process of prion diseases.

  10. Structure and fractal dimension of protein-detergent complexes

    NASA Astrophysics Data System (ADS)

    Chen, Sow-Hsin; Teixeira, José

    1986-11-01

    Small-angle neutron-scattering experiments were made on bovine serum albumin (BSA)-lithium dodecyl sulfate (LDS) complexes in buffer solutions. As increasing amounts of LDS are added, the scattering data indicate that BSA molecules are successively transformed into random coil conformations with LDS forming globular micelles randomly decorating the polypeptide backbones. A cross-section formula is developed which successfully fits small-angle neutron-scattering spectra over the entire Q range. The fractal dimension, the micellar size, and the extent of the denatured protein are simultaneously extracted.

  11. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  12. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix.

    PubMed

    Borlee, Bradley R; Goldman, Aaron D; Murakami, Keiji; Samudrala, Ram; Wozniak, Daniel J; Parsek, Matthew R

    2010-02-01

    Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a beta-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural

  13. Detection of Protein-Protein Interaction Within an RNA-Protein Complex Via Unnatural-Amino-Acid-Mediated Photochemical Crosslinking.

    PubMed

    Yeh, Fu-Lung; Tung, Luh; Chang, Tien-Hsien

    2016-01-01

    Although DExD/H-box proteins are known to unwind RNA duplexes and modulate RNA structures in vitro, it is highly plausible that, in vivo, some may function to remodel RNA-protein complexes. Precisely how the latter is achieved remains a mystery. We investigated this critical issue by using yeast Prp28p, an evolutionarily conserved DExD/H-box splicing factor, as a model system. To probe how Prp28p interacts with spliceosome, we strategically placed p-benzoyl-phenylalanine (BPA), a photoactivatable unnatural amino acid, along the body of Prp28p in vivo. Extracts prepared from these engineered strains were then used to assemble in vitro splicing reactions for BPA-mediated protein-protein crosslinkings. This enabled us, for the first time, to "capture" Prp28p in action. This approach may be applicable to studying the roles of other DExD/H-box proteins functioning in diverse RNA-related pathways, as well as to investigating protein-protein contacts within an RNA-protein complex.

  14. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    SciTech Connect

    Nielsen, Anders Lade

    2009-10-23

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  15. New reagents for increasing ESI multiple charging of proteins and protein complexes.

    PubMed

    Lomeli, Shirley H; Peng, Ivory X; Yin, Sheng; Loo, Rachel R Ogorzalek; Loo, Joseph A

    2010-01-01

    The addition of m-nitrobenzyl alcohol (m-NBA) was shown previously (Lomeli et al., J. Am. Soc. Mass Spectrom. 2009, 20, 593-596) to enhance multiple charging of native proteins and noncovalent protein complexes in electrospray ionization (ESI) mass spectra. Additional new reagents have been found to "supercharge" proteins from nondenaturing solutions; several of these reagents are shown to be more effective than m-NBA for increasing positive charging. Using the myoglobin protein-protoporphyrin IX (heme) complex, the following reagents were shown to increase ESI charging: benzyl alcohol, m-nitroacetophenone, m-nitrobenzonitrile, o-NBA, m-NBA, p-NBA, m-nitrophenyl ethanol, sulfolane (tetramethylene sulfone), and m-(trifluoromethyl)-benzyl alcohol. Based on average charge state, sulfolane displayed a greater charge increase (61%) than m-NBA (21%) for myoglobin in aqueous solutions. The reagents that promote higher ESI charging appear to have low solution-phase basicities and relatively low gas-phase basicities, and are less volatile than water. Another feature of mass spectra from some of the active reagents is that adducts are present on higher charge states, suggesting that a mechanism by which proteins acquire additional charge involves direct interaction with the reagent, in addition to other factors such as surface tension and protein denaturation.

  16. Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

    PubMed Central

    Sudhir, Putty-Reddy; Chen, Chung-Hsuan

    2016-01-01

    A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

  17. In vitro effect of temperature on the conformational structure and collagen binding of SdrF, a Staphylococcus epidermidis adhesin.

    PubMed

    Di Poto, Antonella; Papi, Massimiliano; Trivedi, Sheetal; Maiorana, Alessandro; Gavazzo, Paola; Vassalli, Massimo; Lowy, Franklin D; De Spirito, Marco; Montanaro, Lucio; Imbriani, Marcello; Arciola, Carla Renata; Visai, Livia

    2015-07-01

    Staphylococcus epidermidis is the leading etiologic agent of device-related infections. S. epidermidis is able to bind, by means of the adhesins of its cell wall, the host matrix proteins filming the artificial surfaces. Thence, bacteria cling to biomaterials and infection develops. The effect of temperature on integrity, structure, and biological activity of the collagen-binding adhesin (SdrF) of S. epidermidis has been here investigated. By cloning in E. coli XL1-Blue, a recombinant of the SdrF binding domain B (rSdrFB), carrying an N-terminal polyhistidine, was obtained. Purification was by HiTrap(TM) Chelating HP columns. Assessment of purity, molecular weight, and integrity was by SDS-PAGE. The rSdrFB-collagen binding was investigated by ELISA. A full three-dimensional reconstruction of rSdrFB was achieved by small-angle X-ray scattering (SAXS). At 25 °C, rSdrFB bound to type I collagen in a dose-dependent, saturable manner, with a Kd of 2.48 × 10(-7) M. When temperature increased from 25 to 37 °C, a strong conformational change occurred, together with the abolition of the rSdrFB-collagen binding. The rSdrFB integrity was not affected by temperature variation. SdrFB-collagen binding is switched on/off depending on the temperature. Implications with the infection pathogenesis are enlightened.

  18. Use of Atomic Force Microscopy to Study the Multi-Modular Interaction of Bacterial Adhesins to Mucins

    PubMed Central

    Gunning, A. Patrick; Kavanaugh, Devon; Thursby, Elizabeth; Etzold, Sabrina; MacKenzie, Donald A.; Juge, Nathalie

    2016-01-01

    The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB), a large multi-repeat cell–surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB–MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3), a mammalian lectin characterised by a single carbohydrate binding domain (CRD). The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer. PMID:27834807

  19. Mycobacterium tuberculosis adhesins: potential biomarkers as anti-tuberculosis therapeutic and diagnostic targets.

    PubMed

    Govender, Viveshree S; Ramsugit, Saiyur; Pillay, Manormoney

    2014-09-01

    Adhesion to host cells is a precursor to host colonization and evasion of the host immune response. Conversely, it triggers the induction of the immune response, a process vital to the host's defence against infection. Adhesins are microbial cell surface molecules or structures that mediate the attachment of the microbe to host cells and thus the host-pathogen interaction. They also play a crucial role in bacterial aggregation and biofilm formation. In this review, we discuss the role of adhesins in the pathogenesis of the aetiological agent of tuberculosis, Mycobacterium tuberculosis. We also provide insight into the structure and characteristics of some of the characterized and putative M. tuberculosis adhesins. Finally, we examine the potential of adhesins as targets for the development of tuberculosis control strategies.

  20. Molecular cloning and characterization of the afa-7 and afa-8 gene clusters encoding afimbrial adhesins in Escherichia coli strains associated with diarrhea or septicemia in calves.

    PubMed

    Lalioui, L; Jouve, M; Gounon, P; Le Bouguenec, C

    1999-10-01

    The afa gene clusters, which encode proteins involved in adhesion to epithelial cells, from Escherichia coli strains associated with urinary and intestinal infections in humans have been characterized. Pathogenic isolates of bovine and porcine origin that possess afa-related sequences have recently been described. We report in this work the cloning and characterization of the afa-7 and afa-8 gene clusters from bovine isolates. Hybridization and sequencing experiments revealed that despite similarity in genetic organization, the afa-7 and afa-8 genes, and the well-characterized afa-3 operon expressed by human-pathogenic isolates, correspond to three different members of the afa family of gene clusters. However, like the afa-3 gene cluster, both the afa-7 and afa-8 gene clusters were found to encode an afimbrial adhesin (AfaE) and an invasin (AfaD). The AfaD peptides encoded by the three gene clusters were only 45% identical, but functional complementation experiments indicated that they belong to the same family of invasins. Hemagglutination and adhesion assays demonstrated that the AfaE-VII and AfaE-VIII adhesins bind to different receptors and that these receptors are not the human decay-accelerating factor recognized to be the receptor of all previously described AfaE adhesins. The AfaE-VIII adhesin is very similar to the M agglutinin of human-uropathogenic strains. We used PCR assays to screen 25 bovine strains for afaD and afaE genes of either the afa-7 or afa-8 gene cluster. The afa-8 gene cluster was highly prevalent in bovine isolates previously reported to carry afa-related sequences (23 of 24 strains), particularly in strains producing cytotoxic necrotizing factors (16 of 16 strains). The location of the afa-8 gene cluster on the plasmids or chromosome of these isolates suggests that it could be carried by a mobile element, facilitating its dissemination among bovine-pathogenic E. coli strains.

  1. Small Cofactors May Assist Protein Emergence from RNA World: Clues from RNA-Protein Complexes

    PubMed Central

    Shen, Liang; Ji, Hong-Fang

    2011-01-01

    It is now widely accepted that at an early stage in the evolution of life an RNA world arose, in which RNAs both served as the genetic material and catalyzed diverse biochemical reactions. Then, proteins have gradually replaced RNAs because of their superior catalytic properties in catalysis over time. Therefore, it is important to investigate how primitive functional proteins emerged from RNA world, which can shed light on the evolutionary pathway of life from RNA world to the modern world. In this work, we proposed that the emergence of most primitive functional proteins are assisted by the early primitive nucleotide cofactors, while only a minority are induced directly by RNAs based on the analysis of RNA-protein complexes. Furthermore, the present findings have significant implication for exploring the composition of primitive RNA, i.e., adenine base as principal building blocks. PMID:21789260

  2. Nucleotide sequence of the afimbrial-adhesin-encoding afa-3 gene cluster and its translocation via flanking IS1 insertion sequences.

    PubMed Central

    Garcia, M I; Labigne, A; Le Bouguenec, C

    1994-01-01

    The afa gene clusters encode afimbrial adhesins (AFAs) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains. The plasmid-borne afa-3 gene cluster is responsible for the biosynthesis of the AFA-III adhesin that belongs to the Dr family of hemagglutinins. Reported in this work is the nucleotide sequence of the 9.2-kb insert of the recombinant plasmid pILL61, which contains the afa-3 gene cluster cloned from a cystitis-associated E. coli strain (A30). The afa-3 gene cluster was shown to contain six open reading frames, designated afaA to afaF. It was organized in two divergent transcriptional units. Five of the six Afa products showed marked homologies with proteins encoded by previously described adhesion systems that allowed us to attribute to each of them a putative function in the biogenesis of the AFA-III adhesin. AfaE was identified as the structural adhesin product, whereas AfaB and AfaC were recognized as periplasmic chaperone and outer membrane anchor proteins, respectively. The AfaA and AfaF products were shown to be homologous to the PapI-PapB transcriptional regulatory proteins. No function could be attributed to the AfaD product, the gene of which was previously shown to be dispensable for the synthesis of a functional adhesin. Upstream of the afa-3 gene cluster, a 1.2-kb region was found to be 96% identical to the RepFIB sequence of one of the enterotoxigenic E. coli plasmids (P307), suggesting a common ancestor plasmid. This region contains an integrase-like gene (int). Sequence analysis revealed the presence of an IS1 element between the int gene and the afa-3 gene cluster. Two other IS1 elements were detected and located in the vicinity of the afa-3 gene cluster by hybridization experiments. The afa-3 gene cluster was therefore found to be flanked by two IS1 elements in direct orientation and two in opposite orientations. The afa-3 gene cluster, flanked by two directly oriented IS1 elements, was shown to translocate

  3. Nucleotide sequence of the afimbrial-adhesin-encoding afa-3 gene cluster and its translocation via flanking IS1 insertion sequences.

    PubMed

    Garcia, M I; Labigne, A; Le Bouguenec, C

    1994-12-01

    The afa gene clusters encode afimbrial adhesins (AFAs) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains. The plasmid-borne afa-3 gene cluster is responsible for the biosynthesis of the AFA-III adhesin that belongs to the Dr family of hemagglutinins. Reported in this work is the nucleotide sequence of the 9.2-kb insert of the recombinant plasmid pILL61, which contains the afa-3 gene cluster cloned from a cystitis-associated E. coli strain (A30). The afa-3 gene cluster was shown to contain six open reading frames, designated afaA to afaF. It was organized in two divergent transcriptional units. Five of the six Afa products showed marked homologies with proteins encoded by previously described adhesion systems that allowed us to attribute to each of them a putative function in the biogenesis of the AFA-III adhesin. AfaE was identified as the structural adhesin product, whereas AfaB and AfaC were recognized as periplasmic chaperone and outer membrane anchor proteins, respectively. The AfaA and AfaF products were shown to be homologous to the PapI-PapB transcriptional regulatory proteins. No function could be attributed to the AfaD product, the gene of which was previously shown to be dispensable for the synthesis of a functional adhesin. Upstream of the afa-3 gene cluster, a 1.2-kb region was found to be 96% identical to the RepFIB sequence of one of the enterotoxigenic E. coli plasmids (P307), suggesting a common ancestor plasmid. This region contains an integrase-like gene (int). Sequence analysis revealed the presence of an IS1 element between the int gene and the afa-3 gene cluster. Two other IS1 elements were detected and located in the vicinity of the afa-3 gene cluster by hybridization experiments. The afa-3 gene cluster was therefore found to be flanked by two IS1 elements in direct orientation and two in opposite orientations. The afa-3 gene cluster, flanked by two directly oriented IS1 elements, was shown to translocate

  4. Yeast mitochondrial protein-protein interactions reveal diverse complexes and disease-relevant functional relationships.

    PubMed

    Jin, Ke; Musso, Gabriel; Vlasblom, James; Jessulat, Matthew; Deineko, Viktor; Negroni, Jacopo; Mosca, Roberto; Malty, Ramy; Nguyen-Tran, Diem-Hang; Aoki, Hiroyuki; Minic, Zoran; Freywald, Tanya; Phanse, Sadhna; Xiang, Qian; Freywald, Andrew; Aloy, Patrick; Zhang, Zhaolei; Babu, Mohan

    2015-02-06

    Although detailed, focused, and mechanistic analyses of associations among mitochondrial proteins (MPs) have identified their importance in varied biological processes, a systematic understanding of how MPs function in concert both with one another and with extra-mitochondrial proteins remains incomplete. Consequently, many questions regarding the role of mitochondrial dysfunction in the development of human disease remain unanswered. To address this, we compiled all existing mitochondrial physical interaction data for over 1200 experimentally defined yeast MPs and, through bioinformatic analysis, identified hundreds of heteromeric MP complexes having extensive associations both within and outside the mitochondria. We provide support for these complexes through structure prediction analysis, morphological comparisons of deletion strains, and protein co-immunoprecipitation. The integration of these MP complexes with reported genetic interaction data reveals substantial crosstalk between MPs and non-MPs and identifies novel factors in endoplasmic reticulum-mitochondrial organization, membrane structure, and mitochondrial lipid homeostasis. More than one-third of these MP complexes are conserved in humans, with many containing members linked to clinical pathologies, enabling us to identify genes with putative disease function through guilt-by-association. Although still remaining incomplete, existing mitochondrial interaction data suggests that the relevant molecular machinery is modular, yet highly integrated with non-mitochondrial processes.

  5. Protein-Protein Interaction Investigated by Steered Molecular Dynamics: The TCR-pMHC Complex

    PubMed Central

    Cuendet, Michel A.; Michielin, Olivier

    2008-01-01

    We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the interaction mechanism from a large number of unbinding trajectories by analyzing van der Waals and electrostatic interactions and by computing energy changes in proteins and solvent. In addition, dissociation potentials of mean force are calculated with the Jarzynski identity, using an averaging method developed for our steering scheme. We analyze the convergence of the Jarzynski exponential average, which is hampered by the large amount of dissipative work involved and the complexity of the system. The resulting dissociation free energies largely underestimate experimental values, but the simulations are able to clearly differentiate between wild-type and mutated TCR-pMHC and give insights into the dissociation mechanism. PMID:18621828

  6. Evolution of protein complexity: the blue copper-containing oxidases and related proteins.

    PubMed

    Rydén, L G; Hunt, L T

    1993-01-01

    The blue copper proteins and their relatives have been compared by sequence alignments, by comparison of three-dimensional structures, and by construction of phylogenetic trees. The group contains proteins varying in size from 100 residues to over 2,300 residues in a single chain, containing from zero to nine copper atoms, and with a broad variation in function ranging from electron carrier proteins and oxidases to the blood coagulation factors V and VIII. Difference matrices show the sequence difference to be over 90% for many pairs in the group, yet alignment scores and other evidence suggest that they all evolved from a common ancestor. We have attempted to delineate how this evolution took place and in particular to define the mechanisms by which these proteins acquired an ever-increasing complexity in structure and function. We find evidence for six such mechanisms in this group of proteins: domain enlargement, in which a single domain increases in size from about 100 residues up to 210; domain duplication, which allows for a size increase from about 170 to about 1,000 residues; segment elongation, in which a small segment undergoes multiple successive duplications that can increase the chain size 50-fold; domain recruitment, in which a domain coded elsewhere in the genome is added on to the peptide chain; subunit formation, to form multisubunit proteins; and glycosylation, which in some cases doubles the size of the protein molecule. Size increase allows for the evolution of new catalytic properties, in particular the oxidase function, and for the formation of coagulation factors with multiple interaction sites and regulatory properties. The blood coagulation system is examined as an example in which a system of interacting proteins evolved by successive duplications of larger parts of the genome. The evolution of size, functionality, and diversity is compared with the general question of increase in size and complexity in biology.

  7. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis

    PubMed Central

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S.; Pérez, Amparo Cuéllar; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-01-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  8. CCT complex restricts neuropathogenic protein aggregation via autophagy

    PubMed Central

    Pavel, Mariana; Imarisio, Sara; Menzies, Fiona M.; Jimenez-Sanchez, Maria; Siddiqi, Farah H.; Wu, Xiaoting; Renna, Maurizio; O'Kane, Cahir J.; Crowther, Damian C.; Rubinsztein, David C.

    2016-01-01

    Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC. Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimer's disease. Here, we show that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, our findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity. PMID:27929117

  9. Distribution of adenosine deaminase complexing protein (ADCP) in human tissues.

    PubMed

    Dinjens, W N; ten Kate, J; van der Linden, E P; Wijnen, J T; Khan, P M; Bosman, F T

    1989-12-01

    The normal distribution of adenosine deaminase complexing protein (ADCP) in the human body was investigated quantitatively by ADCP-specific radioimmunoassay (RIA) and qualitatively by immunohistochemistry. In these studies we used a specific rabbit anti-human ADCP antiserum. In all 19 investigated tissues, except erythrocytes, ADCP was found by RIA in the soluble and membrane fractions. From all tissues the membrane fractions contained more ADCP (expressed per mg protein) than the soluble fractions. High membrane ADCP concentrations were found in skin, renal cortex, gastrointestinal tract, and prostate. Immunoperoxidase staining confirmed the predominant membrane-associated localization of the protein. In serous sweat glands, convoluted tubules of renal cortex, bile canaliculi, gastrointestinal tract, lung, pancreas, prostate gland, salivary gland, gallbladder, mammary gland, and uterus, ADCP immunoreactivity was found confined to the luminal membranes of the epithelial cells. These data demonstrate that ADCP is present predominantly in exocrine glands and absorptive epithelia. The localization of ADCP at the secretory or absorptive apex of the cells suggests that the function of ADCP is related to the secretory and/or absorptive process.

  10. The Translocation Domain in Trimeric Autotransporter Adhesins Is Necessary and Sufficient for Trimerization and Autotransportation

    PubMed Central

    Mikula, Kornelia M.; Leo, Jack C.; Łyskowski, Andrzej; Kedracka-Krok, Sylwia; Pirog, Artur

    2012-01-01

    Trimeric autotransporter adhesins (TAAs) comprise one of the secretion pathways of the type V secretion system. The mechanism of their translocation across the outer membrane remains unclear, but it most probably occurs by the formation of a hairpin inside the β-barrel translocation unit, leading to transportation of the passenger domain from the C terminus to the N terminus through the lumen of the β-barrel. We further investigated the phenomenon of autotransportation and the rules that govern it. We showed by coexpressing different Escherichia coli immunoglobulin-binding (Eib) proteins that highly similar TAAs could form stochastically mixed structures (heterotrimers). We further investigated this phenomenon by coexpressing two more distantly related TAAs, EibA and YadA. These, however, did not form heterotrimers; indeed, coexpression was lethal to the cells, leading to elimination of one or another of the genes. However, substituting in either protein the barrel of the other one so that the barrels were identical led to formation of heterotrimers as for Eibs. Our work shows that trimerization of the β-barrel, but not the passenger domain, is necessary and sufficient for TAA secretion while the passenger domain is not. PMID:22155776

  11. Protein-Protein Interactions of the Baculovirus Per Os Infectivity Factors in the PIF Complex.

    PubMed

    Zheng, Qin; Shen, Yunwang; Kon, Xiangshuo; Zhang, Jianjia; Feng, Min; Wu, Xiaofeng

    2017-01-28

    After ingestion of occlusion bodies, the occlusion-derived viruses (ODVs) of baculoviruses establish the first round of infection within the larval host midgut cells. Several ODV envelope proteins, called per os infectivity factors (PIFs), have been shown to be essential for oral infection. Eight PIFs have been identified to date, including P74, PIFs1-6, and Ac110. At least six PIFs: P74, PIFs1-4, PIF6, together with three other ODV-specific proteins: Ac5, P95 (Ac83), and Ac108, have been reported to form a complex on the ODV surface. In this study, in order to understand the interactions of these PIFs, the direct protein-protein interactions of the nine components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex were investigated using yeast two-hybrid (Y2H) combined with bimolecular fluorescence complementation (BiFC) assay. Six direct interactions comprising PIF1-PIF2, PIF1-PIF3, PIF1-PIF4, PIF1-P95, PIF2-PIF3, and PIF3-PIF4, were identified in Y2H analysis, and these results were further verified by BiFC. For P74, PIF6, Ac5 and Ac108, no direct interaction was identified. P95 (Ac83) was identified to interact with PIF1 and further Y2H analysis of the truncations and deletion mutants showed that the predicted P95 chitin-binding domain and PIF1 100-200aa were responsible for P95 interaction with PIF1. Furthermore, a summary of the protein-protein interactions of PIFs reported so far, comprising 10 reciprocal interactions and 2 self-interactions, is presented, which will facilitate our understanding of the characteristic of PIF complex.

  12. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs

    PubMed Central

    Schneider, Tim; Hung, Lee-Hsueh; Schreiner, Silke; Starke, Stefan; Eckhof , Heinrich; Rossbach, Oliver; Reich, Stefan; Medenbach, Jan; Bindereif , Albrecht

    2016-01-01

    Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component. PMID:27510448

  13. Hierarchical structures made of proteins. The complex architecture of spider webs and their constituent silk proteins.

    PubMed

    Heim, Markus; Römer, Lin; Scheibel, Thomas

    2010-01-01

    Biopolymers fulfil a variety of different functions in nature. They conduct various processes inside and outside cells and organisms, with a functionality ranging from storage of information to stabilization, protection, shaping, transport, cellular division, or movement of whole organisms. Within the plethora of biopolymers, the most sophisticated group is of proteinaceous origin: the cytoskeleton of a cell is made of protein filaments that aid in pivotal processes like intracellular transport, movement, and cell division; geckos use a distinct arrangement of keratin-like filaments on their toes which enable them to walk up smooth surfaces, such as walls, and even upside down across ceilings; and spiders spin silks that are extra-corporally used for protection of offspring and construction of complex prey traps. The following tutorial review describes the hierarchical organization of protein fibers, using spider dragline silk as an example. The properties of a dragline silk thread originate from the strictly controlled assembly of the underlying protein chains. The assembly procedure leads to protein fibers showing a complex hierarchical organization comprising three different structural phases. This structural organization is responsible for the outstanding mechanical properties of individual fibers, which out-compete even those of high-performance artificial fibers like Kevlar. Web-weaving spiders produce, in addition to dragline silk, other silks with distinct properties, based on slightly variant constituent proteins--a feature that allows construction of highly sophisticated spider webs with well designed architectures and with optimal mechanical properties for catching prey.

  14. Feature selection and classification of protein-protein complexes based on their binding affinities using machine learning approaches.

    PubMed

    Yugandhar, K; Gromiha, M Michael

    2014-09-01

    Protein-protein interactions are intrinsic to virtually every cellular process. Predicting the binding affinity of protein-protein complexes is one of the challenging problems in computational and molecular biology. In this work, we related sequence features of protein-protein complexes with their binding affinities using machine learning approaches. We set up a database of 185 protein-protein complexes for which the interacting pairs are heterodimers and their experimental binding affinities are available. On the other hand, we have developed a set of 610 features from the sequences of protein complexes and utilized Ranker search method, which is the combination of Attribute evaluator and Ranker method for selecting specific features. We have analyzed several machine learning algorithms to discriminate protein-protein complexes into high and low affinity groups based on their Kd values. Our results showed a 10-fold cross-validation accuracy of 76.1% with the combination of nine features using support vector machines. Further, we observed accuracy of 83.3% on an independent test set of 30 complexes. We suggest that our method would serve as an effective tool for identifying the interacting partners in protein-protein interaction networks and human-pathogen interactions based on the strength of interactions.

  15. Transient Protein-Protein Interaction of the SH3-Peptide Complex via Closely Located Multiple Binding Sites

    PubMed Central

    Hahn, Seungsoo; Kim, Dongsup

    2012-01-01

    Protein-protein interactions play an essential role in cellular processes. Certain proteins form stable complexes with their partner proteins, whereas others function by forming transient complexes. The conventional protein-protein interaction model describes an interaction between two proteins under the assumption that a protein binds to its partner protein through a single binding site. In this study, we improved the conventional interaction model by developing a Multiple-Site (MS) model in which a protein binds to its partner protein through closely located multiple binding sites on a surface of the partner protein by transiently docking at each binding site with individual binding free energies. To test this model, we used the protein-protein interaction mediated by Src homology 3 (SH3) domains. SH3 domains recognize their partners via a weak, transient interaction and are therefore promiscuous in nature. Because the MS model requires large amounts of data compared with the conventional interaction model, we used experimental data from the positionally addressable syntheses of peptides on cellulose membranes (SPOT-synthesis) technique. From the analysis of the experimental data, individual binding free energies for each binding site of peptides were extracted. A comparison of the individual binding free energies from the analysis with those from atomistic force fields gave a correlation coefficient of 0.66. Furthermore, application of the MS model to 10 SH3 domains lowers the prediction error by up to 9% compared with the conventional interaction model. This improvement in prediction originates from a more realistic description of complex formation than the conventional interaction model. The results suggested that, in many cases, SH3 domains increased the protein complex population through multiple binding sites of their partner proteins. Our study indicates that the consideration of general complex formation is important for the accurate description of

  16. Excitation energy transfer in photosynthetic protein-pigment complexes

    NASA Astrophysics Data System (ADS)

    Yeh, Shu-Hao

    Quantum biology is a relatively new research area which investigates the rules that quantum mechanics plays in biology. One of the most intriguing systems in this field is the coherent excitation energy transport (EET) in photosynthesis. In this document I will discuss the theories that are suitable for describing the photosynthetic EET process and the corresponding numerical results on several photosynthetic protein-pigment complexes (PPCs). In some photosynthetic EET processes, because of the electronic coupling between the chromophores within the system is about the same order of magnitude as system-bath coupling (electron-phonon coupling), a non-perturbative method called hierarchy equation of motion (HEOM) is applied to study the EET dynamics. The first part of this thesis includes brief introduction and derivation to the HEOM approach. The second part of this thesis the HEOM method will be applied to investigate the EET process within the B850 ring of the light harvesting complex 2 (LH2) from purple bacteria, Rhodopseudomonas acidophila. The dynamics of the exciton population and coherence will be analyzed under different initial excitation configurations and temperatures. Finally, how HEOM can be implemented to simulate the two-dimensional electronic spectra of photosynthetic PPCs will be discussed. Two-dimensional electronic spectroscopy is a crucial experimental technique to probe EET dynamics in multi-chromophoric systems. The system we are interested in is the 7-chromophore Fenna-Matthews-Olson (FMO) complex from green sulfur bacteria, Prosthecochloris aestuarii. Recent crystallographic studies report the existence of an additional (eighth) chromophore in some of the FMO monomers. By applying HEOM we are able to calculate the two-dimensional electronic spectra of the 7-site and 8-site FMO complexes and investigate the functionality of the eighth chromophore.

  17. The Search Engine for Multi-Proteoform Complexes: An Online Tool for the Identification and Stoichiometry Determination of Protein Complexes.

    PubMed

    Skinner, Owen S; Schachner, Luis F; Kelleher, Neil L

    2016-12-08

    Recent advances in top-down mass spectrometry using native electrospray now enable the analysis of intact protein complexes with relatively small sample amounts in an untargeted mode. Here, we describe how to characterize both homo- and heteropolymeric complexes with high molecular specificity using input data produced by tandem mass spectrometry of whole protein assemblies. The tool described is a "search engine for multi-proteoform complexes," (SEMPC) and is available for free online. The output is a list of candidate multi-proteoform complexes and scoring metrics, which are used to define a distinct set of one or more unique protein subunits, their overall stoichiometry in the intact complex, and their pre- and post-translational modifications. Thus, we present an approach for the identification and characterization of intact protein complexes from native mass spectrometry data. © 2016 by John Wiley & Sons, Inc.

  18. Heterodimeric protein complex identification by naïve Bayes classifiers

    PubMed Central

    2013-01-01

    Background Protein complexes are basic cellular entities that carry out the functions of their components. It can be found that in databases of protein complexes of yeast like CYC2008, the major type of known protein complexes is heterodimeric complexes. Although a number of methods for trying to predict sets of proteins that form arbitrary types of protein complexes simultaneously have been proposed, it can be found that they often fail to predict heterodimeric complexes. Results In this paper, we have designed several features characterizing heterodimeric protein complexes based on genomic data sets, and proposed a supervised-learning method for the prediction of heterodimeric protein complexes. This method learns the parameters of the features, which are embedded in the naïve Bayes classifier. The log-likelihood ratio derived from the naïve Bayes classifier with the parameter values obtained by maximum likelihood estimation gives the score of a given pair of proteins to predict whether the pair is a heterodimeric complex or not. A five-fold cross-validation shows good performance on yeast. The trained classifiers also show higher predictability than various existing algorithms on yeast data sets with approximate and exact matching criteria. Conclusions Heterodimeric protein complex prediction is a rather harder problem than heteromeric protein complex prediction because heterodimeric protein complex is topologically simpler. However, it turns out that by designing features specialized for heterodimeric protein complexes, predictability of them can be improved. Thus, the design of more sophisticate features for heterodimeric protein complexes as well as the accumulation of more accurate and useful genome-wide data sets will lead to higher predictability of heterodimeric protein complexes. Our tool can be downloaded from http://imi.kyushu-u.ac.jp/~om/. PMID:24299017

  19. Weak conservation of structural features in the interfaces of homologous transient protein-protein complexes.

    PubMed

    Sudha, Govindarajan; Singh, Prashant; Swapna, Lakshmipuram S; Srinivasan, Narayanaswamy

    2015-11-01

    Residue types at the interface of protein-protein complexes (PPCs) are known to be reasonably well conserved. However, we show, using a dataset of known 3-D structures of homologous transient PPCs, that the 3-D location of interfacial residues and their interaction patterns are only moderately and poorly conserved, respectively. Another surprising observation is that a residue at the interface that is conserved is not necessarily in the interface in the homolog. Such differences in homologous complexes are manifested by substitution of the residues that are spatially proximal to the conserved residue and structural differences at the interfaces as well as differences in spatial orientations of the interacting proteins. Conservation of interface location and the interaction pattern at the core of the interfaces is higher than at the periphery of the interface patch. Extents of variability of various structural features reported here for homologous transient PPCs are higher than the variation in homologous permanent homomers. Our findings suggest that straightforward extrapolation of interfacial nature and inter-residue interaction patterns from template to target could lead to serious errors in the modeled complex structure. Understanding the evolution of interfaces provides insights to improve comparative modeling of PPC structures.

  20. Atomistic Simulation of Lignocellulosic Biomass and Associated Cellulosomal Protein Complexes

    SciTech Connect

    Petridis, Loukas; Crowley, Michael F; Smith, Jeremy C

    2010-01-01

    Computer simulations have been performed to obtain an atomic-level understanding of lignocellulose structure and the assembly of its associated cellulosomal protein complexes. First, a CHARMM molecular mechanics force field for lignin is derived and validated by performing a molecular dynamics simulation of a crystal of a lignin fragment molecule and comparing simulation-derived structural features with experimental results. Together with the existing force field for polysaccharides, this work provides the basis for full simulations of lignocellulose. Second, the underlying molecular mechanism governing the assembly of various cellulosomal modules is investigated by performing a novel free-energy calculation of the cohesin-dockerin dissociation. Our calculation indicates a free-energy barrier of ~17 kcal/mol and further reveals a stepwise dissociation pathway involving both the central -sheet interface and its adjacent solvent-exposed loop/turn regions clustered at both ends of the -barrel structure.

  1. The complex kinetics of protein folding in wide temperature ranges.

    PubMed

    Wang, Jin

    2004-10-01

    The complex protein folding kinetics in wide temperature ranges is studied through diffusive dynamics on the underlying energy landscape. The well-known kinetic chevron rollover behavior is recovered from the mean first passage time, with the U-shape dependence on temperature. The fastest folding temperature T0 is found to be smaller than the folding transition temperature Tf. We found that the fluctuations of the kinetics through the distribution of first passage time show rather universal behavior, from high-temperature exponential Poissonian kinetics to the relatively low-temperature highly non-exponential kinetics. The transition temperature is at Tk and T0 < Tk < Tf. In certain low-temperature regimes, a power law behavior at long time emerges. At very low temperatures (lower than trapping transition temperature T < T0/(4 approximately 6)), the kinetics is an exponential Poissonian process again.

  2. The EED protein-protein interaction inhibitor A-395 inactivates the PRC2 complex.

    PubMed

    He, Yupeng; Selvaraju, Sujatha; Curtin, Michael L; Jakob, Clarissa G; Zhu, Haizhong; Comess, Kenneth M; Shaw, Bailin; The, Juliana; Lima-Fernandes, Evelyne; Szewczyk, Magdalena M; Cheng, Dong; Klinge, Kelly L; Li, Huan-Qiu; Pliushchev, Marina; Algire, Mikkel A; Maag, David; Guo, Jun; Dietrich, Justin; Panchal, Sanjay C; Petros, Andrew M; Sweis, Ramzi F; Torrent, Maricel; Bigelow, Lance J; Senisterra, Guillermo; Li, Fengling; Kennedy, Steven; Wu, Qin; Osterling, Donald J; Lindley, David J; Gao, Wenqing; Galasinski, Scott; Barsyte-Lovejoy, Dalia; Vedadi, Masoud; Buchanan, Fritz G; Arrowsmith, Cheryl H; Chiang, Gary G; Sun, Chaohong; Pappano, William N

    2017-04-01

    Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

  3. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes

    PubMed Central

    Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

    2016-01-01

    The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains—In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model. PMID:27763556

  4. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes.

    PubMed

    Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

    2016-10-18

    The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains-In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

  5. Retroviral integrase protein and intasome nucleoprotein complex structures

    PubMed Central

    Grawenhoff, Julia; Engelman, Alan N

    2017-01-01

    Retroviral replication proceeds through the integration of a DNA copy of the viral RNA genome into the host cellular genome, a process that is mediated by the viral integrase (IN) protein. IN catalyzes two distinct chemical reactions: 3’-processing, whereby the viral DNA is recessed by a di- or trinucleotide at its 3’-ends, and strand transfer, in which the processed viral DNA ends are inserted into host chromosomal DNA. Although IN has been studied as a recombinant protein since the 1980s, detailed structural understanding of its catalytic functions awaited high resolution structures of functional IN-DNA complexes or intasomes, initially obtained in 2010 for the spumavirus prototype foamy virus (PFV). Since then, two additional retroviral intasome structures, from the α-retrovirus Rous sarcoma virus (RSV) and β-retrovirus mouse mammary tumor virus (MMTV), have emerged. Here, we briefly review the history of IN structural biology prior to the intasome era, and then compare the intasome structures of PFV, MMTV and RSV in detail. Whereas the PFV intasome is characterized by a tetrameric assembly of IN around the viral DNA ends, the newer structures harbor octameric IN assemblies. Although the higher order architectures of MMTV and RSV intasomes differ from that of the PFV intasome, they possess remarkably similar intasomal core structures. Thus, retroviral integration machineries have adapted evolutionarily to utilize disparate IN elements to construct convergent intasome core structures for catalytic function. PMID:28289517

  6. Arabidopsis flower development--of protein complexes, targets, and transport.

    PubMed

    Becker, Annette; Ehlers, Katrin

    2016-03-01

    Tremendous progress has been achieved over the past 25 years or more of research on the molecular mechanisms of floral organ identity, patterning, and development. While collections of floral homeotic mutants of Antirrhinum majus laid the foundation already at the beginning of the previous century, it was the genetic analysis of these mutants in A. majus and Arabidopsis thaliana that led to the development of the ABC model of floral organ identity more than 20 years ago. This intuitive model kick-started research focused on the genetic mechanisms regulating flower development, using mainly A. thaliana as a model plant. In recent years, interactions among floral homeotic proteins have been elucidated, and their direct and indirect target genes are known to a large extent. Here, we provide an overview over the advances in understanding the molecular mechanism orchestrating A. thaliana flower development. We focus on floral homeotic protein complexes, their target genes, evidence for their transport in floral primordia, and how these new results advance our view on the processes downstream of floral organ identity, such as organ boundary formation or floral organ patterning.

  7. Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors*

    PubMed Central

    Cameron, Kate; Najmudin, Shabir; Alves, Victor D.; Bayer, Edward A.; Smith, Steven P.; Bule, Pedro; Waller, Helen; Ferreira, Luís M. A.; Gilbert, Harry J.; Fontes, Carlos M. G. A.

    2015-01-01

    Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼1012 m). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes. PMID:25855788

  8. The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins.

    PubMed Central

    Tchernev, Velizar T.; Mansfield, Traci A.; Giot, Loic; Kumar, A. Madan; Nandabalan, Krishnan; Li, Ying; Mishra, Vishnu S.; Detter, John C.; Rothberg, Jonathan M.; Wallace, Margaret R.; Southwick, Frederick S.; Kingsmore, Stephen F.

    2002-01-01

    BACKGROUND:Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. RESULTS: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). CONCLUSIONS:On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition. PMID:11984006

  9. Solving structures of protein complexes by molecular replacement with Phaser

    SciTech Connect

    McCoy, Airlie J.

    2007-01-01

    Four case studies in using maximum-likelihood molecular replacement, as implemented in the program Phaser, to solve structures of protein complexes are described. Molecular replacement (MR) generally becomes more difficult as the number of components in the asymmetric unit requiring separate MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asymmetric units to be built up from individual components with a ‘tree search with pruning’ approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a number of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large number of copies of the same component in the asymmetric unit or where the components of the asymmetric unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the standard ‘automated MR’ mode and two case studies are used to show how to modify the automated search strategy for problematic cases.

  10. Water Dynamics at Protein-Protein Interfaces: Molecular Dynamics Study of Virus-Host Receptor Complexes.

    PubMed

    Dutta, Priyanka; Botlani, Mohsen; Varma, Sameer

    2014-12-26

    The dynamical properties of water at protein-water interfaces are unlike those in the bulk. Here we utilize molecular dynamics simulations to study water dynamics in interstitial regions between two proteins. We consider two natural protein-protein complexes, one in which the Nipah virus G protein binds to cellular ephrin B2 and the other in which the same G protein binds to ephrin B3. While the two complexes are structurally similar, the two ephrins share only a modest sequence identity of ∼50%. X-ray crystallography also suggests that these interfaces are fairly extensive and contain exceptionally large amounts of waters. We find that while the interstitial waters tend to occupy crystallographic sites, almost all waters exhibit residence times of less than hundred picoseconds in the interstitial region. We also find that while the differences in the sequence of the two ephrins result in quantitative differences in the dynamics of interstitial waters, the trends in the shifts with respect to bulk values are similar. Despite the high wetness of the protein-protein interfaces, the dynamics of interstitial waters are considerably slower compared to the bulk-the interstitial waters diffuse an order of magnitude slower and have 2-3 fold longer hydrogen bond lifetimes and 2-1000 fold slower dipole relaxation rates. To understand the role of interstitial waters, we examine how implicit solvent models compare against explicit solvent models in producing ephrin-induced shifts in the G conformational density. Ephrin-induced shifts in the G conformational density are critical to the allosteric activation of another viral protein that mediates fusion. We find that in comparison with the explicit solvent model, the implicit solvent model predicts a more compact G-B2 interface, presumably because of the absence of discrete waters at the G-B2 interface. Simultaneously, we find that the two models yield strikingly different induced changes in the G conformational density, even

  11. Cooperation of Adhesin Alleles in Salmonella-Host Tropism

    PubMed Central

    De Masi, Leon; Yue, Min; Hu, Changmin; Rakov, Alexey V.; Rankin, Shelley C.

    2017-01-01

    ABSTRACT Allelic combinations and host specificities for three fimbrial adhesins, FimH, BcfD, and StfH, were compared for 262 strains of Salmonella enterica serovar Newport, a frequent human and livestock pathogen. Like FimH, BcfD had two major alleles (designated A and B), whereas StfH had two allelic groups, each with two alleles (subgroup A1 and A2 and subgroup B1 and B2). The most prevalent combinations of FimH/BcfD/StfH alleles in S. Newport were A/A/A1 and B/B/B1. The former set was most frequently found in bovine and porcine strains, whereas the latter combination was most frequently found in environmental and human isolates. Bacteria genetically engineered to express Fim, Bcf, or Stf fimbriae on their surface were tested with the different alleles for binding to human, porcine, and bovine intestinal epithelial cells. The major allelic combinations with bovine and porcine strains (A/A/A1) or with human isolates (B/B/B1) provided at least two alleles capable of binding significantly better than the other alleles to an intestinal epithelial cell line from the respective host(s). However, each combination of alleles kept at least one allele mediating binding to an intestinal epithelial cell from another host. These findings indicated that allelic variation in multiple adhesins of S. Newport contributes to bacterial adaptation to certain preferential hosts without losing the capacity to maintain a broad host range. IMPORTANCE Salmonella enterica remains a leading foodborne bacterial pathogen in the United States; infected livestock serve often as the source of contaminated food products. A study estimated that over a billion Salmonella gastroenteritis cases and up to 33 million typhoid cases occur annually worldwide, with 3.5 million deaths. Although many Salmonella strains with a broad host range present preferential associations with certain host species, it is not clear what determines the various levels of host adaptation. Here, causal properties of host

  12. Catch-bond mechanism of the bacterial adhesin FimH.

    PubMed

    Sauer, Maximilian M; Jakob, Roman P; Eras, Jonathan; Baday, Sefer; Eriş, Deniz; Navarra, Giulio; Bernèche, Simon; Ernst, Beat; Maier, Timm; Glockshuber, Rudi

    2016-03-07

    Ligand-receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell-cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force.

  13. sae is essential for expression of the staphylococcal adhesins Eap and Emp.

    PubMed

    Harraghy, Niamh; Kormanec, Jan; Wolz, Christiane; Homerova, Dagmar; Goerke, Christiane; Ohlsen, Knut; Qazi, Saara; Hill, Philip; Herrmann, Mathias

    2005-06-01

    Eap and Emp are two Staphylococcus aureus adhesins initially described as extracellular matrix binding proteins. Eap has since emerged as being important in adherence to and invasion of eukaryotic cells, as well as being described as an immunomodulator and virulence factor in chronic infections. This paper describes the mapping of the transcription start point of the eap and emp promoters. Moreover, using reporter-gene assays and real-time PCR in defined regulatory mutants, environmental conditions and global regulators affecting expression of eap and emp were investigated. Marked differences were found in expression of eap and emp between strain Newman and the 8325 derivatives SH1000 and 8325-4. Moreover, both genes were repressed in the presence of glucose. Analysis of expression of both genes in various regulatory mutants revealed that sarA and agr were involved in their regulation, but the data suggested that there were additional regulators of both genes. In a sae mutant, expression of both genes was severely repressed. sae expression was also reduced in the presence of glucose, suggesting that repression of eap and emp in glucose-containing medium may, in part, be a consequence of a decrease in expression of sae.

  14. Catch-bond mechanism of the bacterial adhesin FimH

    PubMed Central

    Sauer, Maximilian M.; Jakob, Roman P.; Eras, Jonathan; Baday, Sefer; Eriş, Deniz; Navarra, Giulio; Bernèche, Simon; Ernst, Beat; Maier, Timm; Glockshuber, Rudi

    2016-01-01

    Ligand–receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell–cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force. PMID:26948702

  15. Using contrast patterns between true complexes and random subgraphs in PPI networks to predict unknown protein complexes

    PubMed Central

    Liu, Quanzhong; Song, Jiangning; Li, Jinyan

    2016-01-01

    Most protein complex detection methods utilize unsupervised techniques to cluster densely connected nodes in a protein-protein interaction (PPI) network, in spite of the fact that many true complexes are not dense subgraphs. Supervised methods have been proposed recently, but they do not answer why a group of proteins are predicted as a complex, and they have not investigated how to detect new complexes of one species by training the model on the PPI data of another species. We propose a novel supervised method to address these issues. The key idea is to discover emerging patterns (EPs), a type of contrast pattern, which can clearly distinguish true complexes from random subgraphs in a PPI network. An integrative score of EPs is defined to measure how likely a subgraph of proteins can form a complex. New complexes thus can grow from our seed proteins by iteratively updating this score. The performance of our method is tested on eight benchmark PPI datasets and compared with seven unsupervised methods, two supervised and one semi-supervised methods under five standards to assess the quality of the predicted complexes. The results show that in most cases our method achieved a better performance, sometimes significantly. PMID:26868667

  16. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes

    PubMed Central

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  17. 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

  18. The urease enzyme of Helicobacter pylori does not function as an adhesin.

    PubMed

    Clyne, M; Drumm, B

    1996-07-01

    Helicobacter pylori urease is essential for colonization of the gastric mucosa irrespective of whether the stomach is acidic or hypochlorhydric. It has therefore been speculated that the enzyme functions as an adhesin. The aim of this study was to compare the adherence of H. pylori N6 with the adherence of an isogenic urease-negative mutant, strain N6(ureB::TnKm), to gastric cells. Strain N6 originated from a patient with gastritis. Strain N6(ureB::TnKm) is specifically modified in the gene which encodes the large subunit of urease, UreB, and hence does not form a UreA-UreB enzyme complex. We have used flow cytometry to assess the adherence of H. pylori to the cells. We have also used phase-contrast microscopy to assess the adherence of the organism to Kato III cells. In the absence of urea both strains bound to Kato III cells and to primary gastric cells. Binding of both strains to the cells occurred rapidly. The presence of urea in the incubation medium decreased the binding of strain N6 to the cells. This was due to a rise in the pH of the incubation medium, which caused loss of viability of the organism. Urea had no effect on the adherence of strain N6(ureB::TnKm). We conclude that the urease of H. pylori does not play a role in the adherence of the organism to gastric cells.

  19. XacFhaB adhesin, an important Xanthomonas citri ssp. citri virulence factor, is recognized as a pathogen-associated molecular pattern.

    PubMed

    Garavaglia, Betiana S; Zimaro, Tamara; Abriata, Luciano A; Ottado, Jorgelina; Gottig, Natalia

    2016-12-01

    Adhesion to host tissue is one of the key steps of the bacterial pathogenic process. Xanthomonas citri ssp. citri possesses a non-fimbrial adhesin protein, XacFhaB, required for bacterial attachment, which we have previously demonstrated to be an important virulence factor for the development of citrus canker. XacFhaB is a 4753-residue-long protein with a predicted β-helical fold structure, involved in bacterial aggregation, biofilm formation and adhesion to the host. In this work, to further characterize this protein and considering its large size, XacFhaB was dissected into three regions based on bioinformatic and structural analyses for functional studies. First, the capacity of these protein regions to aggregate bacterial cells was analysed. Two of these regions were able to form bacterial aggregates, with the most amino-terminal region being dispensable for this activity. Moreover, XacFhaB shows features resembling pathogen-associated molecular patterns (PAMPs), which are recognized by plants. As PAMPs activate plant basal immune responses, the role of the three XacFhaB regions as elicitors of these responses was investigated. All adhesin regions were able to induce basal immune responses in host and non-host plants, with a stronger activation by the carboxyl-terminal region. Furthermore, pre-infiltration of citrus leaves with XacFhaB regions impaired X. citri ssp. citri growth, confirming the induction of defence responses and restraint of citrus canker. This work reveals that adhesins from plant pathogens trigger plant defence responses, opening up new pathways for the development of protective strategies for disease control.

  20. The Staphylococcal Biofilm: Adhesins, regulation, and host response

    PubMed Central

    Paharik, Alexandra E.; Horswill, Alexander R.

    2015-01-01

    The Staphylococci comprise a diverse genus of Gram-positive, non-motile commensal organisms that inhabit the skin and mucous membranes of humans and other mammals. In general, Staphylococci are benign members of the natural flora, but many species have the capacity to be opportunistic pathogens, mainly infecting individuals who have medical device implants or are otherwise immunocompromised. S. aureus and S. epidermidis are a major source of hospital-acquired infections and are the most common causes of surgical site infections and central line-associated bloodstream infections. The ability of Staphylococci to form biofilms in vivo makes them highly resistant to chemotherapeutics and leads to chronic diseases. These biofilm infections include osteomyelitis, endocarditis, medical device implants, and persistence in the cystic fibrosis lung. Here, we provide a comprehensive analysis of our current understanding of Staphylococcal biofilm formation, with an emphasis on adhesins and regulation, while also addressing how Staphylococcal biofilms interact with the immune system. On the whole, this review will provide a thorough picture of biofilm formation of the Staphylococcus genus and how this mode of growth impacts the host. PMID:27227309

  1. Sticky Matrix: Adhesion Mechanism of the Staphylococcal Polysaccharide Intercellular Adhesin.

    PubMed

    Formosa-Dague, Cécile; Feuillie, Cécile; Beaussart, Audrey; Derclaye, Sylvie; Kucharíková, Soňa; Lasa, Iñigo; Van Dijck, Patrick; Dufrêne, Yves F

    2016-03-22

    The development of bacterial biofilms on surfaces leads to hospital-acquired infections that are difficult to fight. In Staphylococci, the cationic polysaccharide intercellular adhesin (PIA) forms an extracellular matrix that connects the cells together during biofilm formation, but the molecular forces involved are unknown. Here, we use advanced force nanoscopy techniques to unravel the mechanism of PIA-mediated adhesion in a clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strain. Nanoscale multiparametric imaging of the structure, adhesion, and elasticity of bacteria expressing PIA shows that the cells are surrounded by a soft and adhesive matrix of extracellular polymers. Cell surface softness and adhesion are dramatically reduced in mutant cells deficient for the synthesis of PIA or under unfavorable growth conditions. Single-cell force spectroscopy demonstrates that PIA promotes cell-cell adhesion via the multivalent electrostatic interaction with polyanionic teichoic acids on the S. aureus cell surface. This binding mechanism rationalizes, at the nanoscale, the well-known ability of PIA to strengthen intercellular adhesion in staphylococcal biofilms. Force nanoscopy offers promising prospects for understanding the fundamental forces in antibiotic-resistant biofilms and for designing anti-adhesion compounds targeting matrix polymers.

  2. Methods for protein complex prediction and their contributions towards understanding the organisation, function and dynamics of complexes.

    PubMed

    Srihari, Sriganesh; Yong, Chern Han; Patil, Ashwini; Wong, Limsoon

    2015-09-14

    Complexes of physically interacting proteins constitute fundamental functional units responsible for driving biological processes within cells. A faithful reconstruction of the entire set of complexes is therefore essential to understand the functional organisation of cells. In this review, we discuss the key contributions of computational methods developed till date (approximately between 2003 and 2015) for identifying complexes from the network of interacting proteins (PPI network). We evaluate in depth the performance of these methods on PPI datasets from yeast, and highlight their limitations and challenges, in particular at detecting sparse and small or sub-complexes and discerning overlapping complexes. We describe methods for integrating diverse information including expression profiles and 3D structures of proteins with PPI networks to understand the dynamics of complex formation, for instance, of time-based assembly of complex subunits and formation of fuzzy complexes from intrinsically disordered proteins. Finally, we discuss methods for identifying dysfunctional complexes in human diseases, an application that is proving invaluable to understand disease mechanisms and to discover novel therapeutic targets. We hope this review aptly commemorates a decade of research on computational prediction of complexes and constitutes a valuable reference for further advancements in this exciting area.

  3. Assembly of Photosynthetic Antenna Protein / Pigments Complexes from Algae and Plants for Development of Nanobiodevices

    DTIC Science & Technology

    2012-07-10

    Assembly of Photosynthetic Antenna Protein / Pigments Complexes from Algae and Plants for Development of Nanobiodevices Key...Assembly of Photosynthetic Antenna Protein / Pigments Complexes from Algae and Plants for Development of Nanobiodevices 5a. CONTRACT NUMBER...unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT This is the report of a project to use photosynthetic antenna pigment complexes from algae and plants as

  4. Sampling small-scale and large-scale conformational changes in proteins and molecular complexes

    NASA Astrophysics Data System (ADS)

    Yun, Mi-Ran; Mousseau, N.; Derreumaux, P.

    2007-03-01

    Sampling of small-scale and large-scale motions is important in various computational tasks, such as protein-protein docking and ligand binding. Here, we report further development and applications of the activation-relaxation technique for internal coordinate space trajectories (ARTIST). This method generates conformational moves of any complexity and size by identifying and crossing well-defined saddle points connecting energy minima. Simulations on two all-atom proteins and three protein complexes containing between 70 and 300 amino acids indicate that ARTIST opens the door to the full treatment of all degrees of freedom in dense systems such as protein-protein complexes.

  5. Synaptonemal Complex Protein 3 Transcript Analysis in Breast Cancer

    PubMed Central

    MOBASHERI, Maryam Beigom; SHIRKOOHI, Reza; MODARRESSI, Mohammad Hossein

    2016-01-01

    Background: Breast cancer is the most frequent cancer in women. Cancer/Testis antigens are immunogenic proteins ectopically expressed in human neoplasms. Synaptonemal complex protein 3 (SYCP3) belongs to cancer/testis genes family involved in meiotic events and spermatogenesis. The aim of this study was to express analysis of SYCP3 in breast cancer and validate it as a breast cancer biomarker. Methods: Expression of SYCP3 transcripts in 47 breast tumors, 6 breast cancer cell lines (MCF7, SKBR3, T47D, BT474, MDA-MB-231 and MDA-MB 468), 5 normal breast and 2 testis tissues was studied by Real Time RT-PCR reaction. The reference genes phosphoglucomutase 1 and hypoxanthine guanine phosphoribosyl transferase were used as reactions normalizers. The software tool REST 2009 was applied for statistical analysis of the data. The research was conducted from Apr 2014 to August 2015 in Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Results: All of the studied breast cancer cell lines showed very high levels of SYCP3 overexpression in comparison to normal breast (P=0.001) and even to normal testis (P=0.001), except for MCF7 cell line. Breast tumors showed moderately increasing in transcript changes in comparison to normal breast. Conclusion: SYCP3 is a known testis-specific gene, but interestingly five out of six studied breast cancer of cell lines showed higher expression levels of SYCP3 in comparison to normal testis and normal breast tissues. SYCP3 has critical role in cell division with known interaction with the tumor suppressor genes, BRCA1 and BRCA2, which are critical genes in breast cancer. PMID:28053928

  6. Targeting a dynamic protein-protein interaction: fragment screening against the malaria myosin A motor complex.

    PubMed

    Douse, Christopher H; Vrielink, Nina; Wenlin, Zhang; Cota, Ernesto; Tate, Edward W

    2015-01-01

    Motility is a vital feature of the complex life cycle of Plasmodium falciparum, the apicomplexan parasite that causes human malaria. Processes such as host cell invasion are thought to be powered by a conserved actomyosin motor (containing myosin A or myoA), correct localization of which is dependent on a tight interaction with myosin A tail domain interacting protein (MTIP) at the inner membrane of the parasite. Although disruption of this protein-protein interaction represents an attractive means to investigate the putative roles of myoA-based motility and to inhibit the parasitic life cycle, no small molecules have been identified that bind to MTIP. Furthermore, it has not been possible to obtain a crystal structure of the free protein, which is highly dynamic and unstable in the absence of its natural myoA tail partner. Herein we report the de novo identification of the first molecules that bind to and stabilize MTIP via a fragment-based, integrated biophysical approach and structural investigations to examine the binding modes of hit compounds. The challenges of targeting such a dynamic system with traditional fragment screening workflows are addressed throughout.

  7. Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus.

    PubMed

    Villareal, Valerie A; Spirig, Thomas; Robson, Scott A; Liu, Mengyao; Lei, Benfang; Clubb, Robert T

    2011-09-14

    Iron is an essential nutrient for the bacterial pathogen Staphylococcus aureus . Heme in hemoglobin (Hb) is the most abundant source of iron in the human body and during infections is captured by S. aureus using iron-regulated surface determinant (Isd) proteins. A central step in this process is the transfer of heme between the cell wall associated IsdA and IsdC hemoproteins. Biochemical evidence indicates that heme is transferred via an activated IsdA:heme:IsdC heme complex. Transfer is rapid and occurs up to 70,000 times faster than indirect mechanisms in which heme is released into the solvent. To gain insight into the mechanism of transfer, we modeled the structure of the complex using NMR paramagnetic relaxation enhancement (PRE) methods. Our results indicate that IsdA and IsdC transfer heme via an ultraweak affinity "handclasp" complex that juxtaposes their respective 3(10) helices and β7/β8 loops. Interestingly, PRE also identified a set of transient complexes that could represent high-energy pre-equilibrium encounter species that form prior to the stereospecific handclasp complex. Targeted amino acid mutagenesis and stopped-flow measurements substantiate the functional relevance of a PRE-derived model, as mutation of interfacial side chains significantly slows the rate of transfer. IsdA and IsdC bind heme using NEAr Transporter (NEAT) domains that are conserved in many species of pathogenic Gram-positive bacteria. Heme transfer in these microbes may also occur through structurally similar transient stereospecific complexes.

  8. High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

    PubMed

    Walian, Peter J; Allen, Simon; Shatsky, Maxim; Zeng, Lucy; Szakal, Evelin D; Liu, Haichuan; Hall, Steven C; Fisher, Susan J; Lam, Bonita R; Singer, Mary E; Geller, Jil T; Brenner, Steven E; Chandonia, John-Marc; Hazen, Terry C; Witkowska, H Ewa; Biggin, Mark D; Jap, Bing K

    2012-12-07

    Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.

  9. PCE-FR: A Novel Method for Identifying Overlapping Protein Complexes in Weighted Protein-Protein Interaction Networks Using Pseudo-Clique Extension Based on Fuzzy Relation.

    PubMed

    Cao, Buwen; Luo, Jiawei; Liang, Cheng; Wang, Shulin; Ding, Pingjian

    2016-10-01

    Identifying overlapping protein complexes in protein-protein interaction (PPI) networks can provide insight into cellular functional organization and thus elucidate underlying cellular mechanisms. Recently, various algorithms for protein complexes detection have been developed for PPI networks. However, majority of algorithms primarily depend on network topological feature and/or gene expression profile, failing to consider the inherent biological meanings between protein pairs. In this paper, we propose a novel method to detect protein complexes using pseudo-clique extension based on fuzzy relation (PCE-FR). Our algorithm operates in three stages: it first forms the nonoverlapping protein substructure based on fuzzy relation and then expands each substructure by adding neighbor proteins to maximize the cohesive score. Finally, highly overlapped candidate protein complexes are merged to form the final protein complex set. Particularly, our algorithm employs the biological significance hidden in protein pairs to construct edge weight for protein interaction networks. The experiment results show that our method can not only outperform classical algorithms such as CFinder, ClusterONE, CMC, RRW, HC-PIN, and ProRank +, but also achieve ideal overall performance in most of the yeast PPI datasets in terms of composite score consisting of precision, accuracy, and separation. We further apply our method to a human PPI network from the HPRD dataset and demonstrate it is very effective in detecting protein complexes compared to other algorithms.

  10. Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection.

    PubMed

    Deuschle, Eva; Keller, Birgit; Siegfried, Alexandra; Manncke, Birgit; Spaeth, Tanja; Köberle, Martin; Drechsler-Hake, Doreen; Reber, Julia; Böttcher, Ralph T; Autenrieth, Stella E; Autenrieth, Ingo B; Bohn, Erwin; Schütz, Monika

    2016-02-01

    Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors.

  11. Serine-Rich Repeat Adhesins Contribute to Streptococcus gordonii-Induced Maturation of Human Dendritic Cells

    PubMed Central

    Ko, Eun Byeol; Kim, Sun Kyung; Seo, Ho Seong; Yun, Cheol-Heui; Han, Seung Hyun

    2017-01-01

    Dendritic cells (DCs) play a pivotal role in the induction of immunity by recognition, capture, process, and presentation of antigens from infectious microbes. Streptococcus gordonii is able to cause life-threatening systemic diseases such as infective endocarditis. Serine-rich repeat (SRR) glycoproteins of S. gordonii are sialic acid-binding adhesins mediating the bacterial adherence to the host and the development of infective endocarditis. Thus, the SRR adhesins are potentially involved in the bacterial adherence to DCs and the maturation and activation of DCs required for the induction of immunity to S. gordonii. Here, we investigated the phenotypic and functional changes of human monocyte-derived DCs treated with wild-type S. gordonii or the SRR adhesin-deficient mutant. The mutant poorly bound to DCs and only weakly increased the expression of CD83, CD86, MHC class II, and PD-L1 on DCs compared with the wild-type. In addition, the mutant induced lower levels of TNF-α, IL-6, and IL-12 than the wild-type in DCs. When DCs sensitized with the mutant were co-cultured with autologous T cells, they induced weaker proliferation and activation of T cells than DCs stimulated with the wild-type. Blockade of SRR adhesin with 3′-sialyllactose markedly reduced S. gordonii binding and internalization, causing attenuation of the bacterial immunostimulatory potency in DC maturation. Collectively, our results suggest that SRR adhesins of S. gordonii are important for maturation and activation of DCs.

  12. HAMLET - A protein-lipid complex with broad tumoricidal activity.

    PubMed

    Ho, James C S; Nadeem, Aftab; Svanborg, Catharina

    2017-01-15

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential.

  13. Adenosine deaminase complexing protein (ADCP) immunoreactivity in colorectal adenocarcinoma.

    PubMed

    ten Kate, J; van den Ingh, H F; Khan, P M; Bosman, F T

    1986-04-15

    Immunoreactive adenosine deaminase complexing protein (ADCP) was studied in 91 human colorectal adenocarcinomas. The expression of ADCP was correlated with that of secretory component (SC) and carcinoembryonic antigen (CEA), with the histological grade and the Dukes' stage of the carcinomas. The histological grade was scored semi-quantitatively according to 5 structural and 4 cytological variables. ADCP expression was observed in 3 different staining patterns, namely: (1) diffuse cytoplasmic (77% of the carcinomas); (2) granular cytoplasmic (13%); and (3) membrane-associated (66%). These patterns were observed alone or in combination. Eleven percent of the carcinomas exhibited no ADCP immunoreactivity. Linear regression analysis showed that the expression of ADCP correlates with that of SC and CEA. However, no significant correlation emerged between the histological parameters or the Dukes' stage and any of the immunohistological parameters. Comparison of the histological characteristics of carcinomas exhibiting little or no ADCP immunoreactivity with those showing extensive immunoreactivity, showed that membranous ADCP immunoreactivity occurs more frequently in well-differentiated carcinomas. Structural parameters showed a better correlation with membranous ADCP expression than the cytological variables. It is concluded that membranous expression of ADCP and CEA are indicators of a high level of differentiation as reflected primarily in the structural characteristics of the tumor.

  14. Stability and Immunogenicity of Hypoallergenic Peanut Protein-Polyphenol Complexes During In Vitro Pepsin Digestion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Allergenic peanut proteins are relatively resistant to digestion, and if digested, metabolized peptides tend to remain large and immunoreactive, triggering allergic reactions in sensitive individuals. In this study, the stability of hypoallergenic peanut protein-polyphenol complexes was evaluated d...

  15. Design and construction of self-assembling supramolecular protein complexes using artificial and fusion proteins as nanoscale building blocks.

    PubMed

    Kobayashi, Naoya; Arai, Ryoichi

    2017-02-01

    The central goal of nanobiotechnology is to design and construct novel biomaterials of nanometer sizes. In this short review, we describe recent progress of several approaches for designing and creating artificial self-assembling protein complexes and primarily focus on the following biotechnological strategies for using artificial and fusion proteins as nanoscale building blocks: fusion proteins designed for symmetrical self-assembly; three-dimensional domain-swapped oligomers; self-assembling designed coiled-coil peptide modules; metal-directed self-assembling engineered proteins; computationally designed self-assembling de novo proteins; and self-assembling protein nanobuilding blocks (PN-Blocks) using an intermolecularly folded dimeric de novo protein. These state-of-the-art nanobiotechnologies for designing supramolecular protein complexes will facilitate the development of novel functional nanobiomaterials.

  16. Dra/AfaE adhesin of uropathogenic Dr/Afa+ Escherichia coli mediates mortality in pregnant rats.

    PubMed

    Wroblewska-Seniuk, K; Selvarangan, R; Hart, A; Pladzyk, R; Goluszko, P; Jafari, A; du Merle, L; Nowicki, S; Yallampalli, C; Le Bouguénec, C; Nowicki, B

    2005-11-01

    Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa+ E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE+ afaD and afaE afaD+ mutants. The clinical E. coli strain (afaE+ afaD+) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE+ afaD+ strain, followed by the group infected with the afaE+ afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE+ strains were the most invasive (afaE+ afaD strain > afaE+ afaD+ strain), while the afaE mutant strains (afaE afaD+ and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE+ E. coli strains in vitro.

  17. Dra/AfaE Adhesin of Uropathogenic Dr/Afa+ Escherichia coli Mediates Mortality in Pregnant Rats

    PubMed Central

    Wroblewska-Seniuk, K.; Selvarangan, R.; Hart, A.; Pladzyk, R.; Goluszko, P.; Jafari, A.; du Merle, L.; Nowicki, S.; Yallampalli, C.; Le Bouguénec, C.; Nowicki, B.

    2005-01-01

    Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa+ E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE+ afaD and afaE afaD+ mutants. The clinical E. coli strain (afaE+ afaD+) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE+ afaD+ strain, followed by the group infected with the afaE+ afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE+ strains were the most invasive (afaE+ afaD strain > afaE+ afaD+ strain), while the afaE mutant strains (afaE afaD+ and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE+ E. coli strains in vitro. PMID:16239563

  18. Proteomic analysis of exported chaperone/co-chaperone complexes of P. falciparum reveals an array of complex protein-protein interactions

    PubMed Central

    Zhang, Qi; Ma, Cheng; Oberli, Alexander; Zinz, Astrid; Engels, Sonja; Przyborski, Jude M.

    2017-01-01

    Malaria parasites modify their human host cell, the mature erythrocyte. This modification is mediated by a large number of parasite proteins that are exported to the host cell, and is also the underlying cause for the pathology caused by malaria infection. Amongst these proteins are many Hsp40 co-chaperones, and a single Hsp70. These proteins have been implicated in several processes in the host cell, including a potential role in protein transport, however the further molecular players in this process remain obscure. To address this, we have utilized chemical cross-linking followed by mass spectrometry and immunoblotting to isolate and characterize proteins complexes containing an exported Hsp40 (PFE55), and the only known exported Hsp70 (PfHsp70x). Our data reveal that both of these proteins are contained in high molecular weight protein complexes. These complexes are found both in the infected erythrocyte, and within the parasite-derived compartment referred to as the parasitophorous vacuole. Surprisingly, our data also reveal an association of PfHsp70x with components of PTEX, a putative protein translocon within the membrane of the parasitophorous vacuole. Our results suggest that the P. falciparum- infected human erythrocyte contains numerous high molecular weight protein complexes, which may potentially be involved in host cell modification. PMID:28218284

  19. A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen.

    PubMed

    Nallapareddy, Sreedhar R; Singh, Kavindra V; Okhuysen, Pablo C; Murray, Barbara E

    2008-09-01

    Enterococcus faecium recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some E. faecium strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90 E. faecium isolates (99% acm(+)) and found that the Acm protein was detected predominantly in clinically derived isolates, while the acm gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (P complex 17 (CC17), which has emerged globally. Taken together, our findings support the hypothesis that Acm has contributed to the emergence of E. faecium and CC17 in nosocomial infections.

  20. Biotin-Streptavidin Affinity Purification of RNA-Protein Complexes Assembled In Vitro.

    PubMed

    Hou, Shuai; Shi, Lei; Lei, Haixin

    2016-01-01

    RNA-protein complexes are essential for the function of different RNAs, yet purification of specific RNA-protein complexes can be complicated and is a major obstacle in understanding the mechanism of regulatory RNAs. Here we present a protocol to purify RNA-protein complexes assembled in vitro based on biotin-streptavidin affinity. In vitro transcribed RNA is labeled with (32)P and biotin, ribonucleoprotein particles or RNPs are assembled by incubation of RNA in nuclear extract and fractionated using gel filtration, and RNP fractions are pooled for biotin-streptavidin affinity purification. The amount of RNA-protein complexes purified following this protocol is sufficient for mass spectrometry.

  1. Qualitative and Quantitative Protein Complex Prediction Through Proteome-Wide Simulations.

    PubMed

    Rizzetto, Simone; Priami, Corrado; Csikász-Nagy, Attila

    2015-10-01

    Despite recent progress in proteomics most protein complexes are still unknown. Identification of these complexes will help us understand cellular regulatory mechanisms and support development of new drugs. Therefore it is really important to establish detailed information about the composition and the abundance of protein complexes but existing algorithms can only give qualitative predictions. Herein, we propose a new approach based on stochastic simulations of protein complex formation that integrates multi-source data--such as protein abundances, domain-domain interactions and functional annotations--to predict alternative forms of protein complexes together with their abundances. This method, called SiComPre (Simulation based Complex Prediction), achieves better qualitative prediction of yeast and human protein complexes than existing methods and is the first to predict protein complex abundances. Furthermore, we show that SiComPre can be used to predict complexome changes upon drug treatment with the example of bortezomib. SiComPre is the first method to produce quantitative predictions on the abundance of molecular complexes while performing the best qualitative predictions. With new data on tissue specific protein complexes becoming available SiComPre will be able to predict qualitative and quantitative differences in the complexome in various tissue types and under various conditions.

  2. Stabilization of a binary protein complex by intein-mediated cyclization.

    PubMed

    Jeffries, Cy M; Graham, Stephen C; Stokes, Philippa H; Collyer, Charles A; Guss, J Mitchell; Matthews, Jacqueline M

    2006-11-01

    The study of protein-protein interactions can be hampered by the instability of one or more of the protein complex components. In this study, we showed that intein-mediated cyclization can be used to engineer an artificial intramolecular cyclic protein complex between two interacting proteins: the largely unstable LIM-only protein 4 (LMO4) and an unstructured domain of LIM domain binding protein 1 (ldb1). The X-ray structure of the cyclic complex is identical to noncyclized versions of the complex. Chemical and thermal denaturation assays using intrinsic tryptophan fluorescence and dynamic light scattering were used to compare the relative stabilities of the cyclized complex, the intermolecular (or free) complex, and two linear versions of the intramolecular complex (in which the interacting domains of LMO4 and ldb1 were fused, via a flexible linker, in either orientation). In terms of resistance to denaturation, the cyclic complex is the most stable variant and the intermolecular complex is the least stable; however, the two linear intramolecular variants show significant differences in stability. These differences appear to be related to the relative contact order (the average distance in sequence between residues that make contacts within a structure) of key binding residues at the interface of the two proteins. Thus, the restriction of the more stable component of a complex may enhance stability to a greater extent than restraining less stable components.

  3. High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

    PubMed Central

    2012-01-01

    Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

  4. Site specific chemoselective labelling of proteins with robust and highly sensitive Ru(II) bathophenanthroline complexes.

    PubMed

    Uzagare, Matthew C; Claussnitzer, Iris; Gerrits, Michael; Bannwarth, Willi

    2012-03-21

    The bioorthogonal and chemoselective fluorescence labelling of several cell-free synthesized proteins containing a site-specifically incorporated azido amino acid was possible using different alkyne-functionalized Ru(II) bathophenanthroline complexes. We were able to achieve a selective labelling even in complex mixtures of proteins despite the fact that ruthenium dyes normally show a high tendency for unspecific interactions with proteins and are commonly used for total staining of proteins. Since the employed Ru complexes are extremely robust, photo-stable and highly sensitive, the approach should be applicable to the production of labelled proteins for single molecule spectroscopy and fluorescence-based interaction studies.

  5. Affinity proteomics to study endogenous protein complexes: Pointers, pitfalls, preferences and perspectives

    PubMed Central

    LaCava, John; Molloy, Kelly R.; Taylor, Martin S.; Domanski, Michal; Chait, Brian T.; Rout, Michael P.

    2015-01-01

    Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls. PMID:25757543

  6. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    PubMed

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

  7. Immunogenicity of a prototype enterotoxigenic Escherichia coli adhesin vaccine in mice and nonhuman primates.

    PubMed

    Sincock, Stephanie A; Hall, Eric R; Woods, Colleen M; O'Dowd, Aisling; Poole, Steven T; McVeigh, Annette L; Nunez, Gladys; Espinoza, Nereyda; Miller, Milagros; Savarino, Stephen J

    2016-01-04

    Enterotoxigenic Escherichia coli (ETEC) are the most common cause of bacterial diarrhea in young children in developing countries and in travelers. Efforts to develop an ETEC vaccine have intensified in the past decade, and intestinal colonization factors (CFs) are somatic components of most investigational vaccines. CFA/I and related Class 5 fimbrial CFs feature a major stalk-forming subunit and a minor, antigenically conserved tip adhesin. We hypothesized that the tip adhesin is critical for stimulating antibodies that specifically inhibit ETEC attachment to the small intestine. To address this, we compared the capacity of donor strand complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, and CFA/I fimbriae to elicit anti-adhesive antibodies in mice, using hemagglutination inhibition (HAI) as proxy for neutralization of intestinal adhesion. When given with genetically attenuated heat-labile enterotoxin LTR192G as adjuvant by intranasal (IN) or orogastric (OG) vaccination, dscCfaE exceeded CFA/I fimbriae in eliciting serum HAI titers and anti-CfaE antibody titers. Based on these findings, we vaccinated Aotus nancymaae nonhuman primates (NHP) with dscCfaE alone or admixed with one of two adjuvants, LTR192G and cholera toxin B-subunit, by IN and OG administration. Only IN vaccination with dscCfaE with either adjuvant elicited substantial serum HAI titers and IgA and IgG anti-adhesin responses, with the latter detectable a year after vaccination. In conclusion, we have shown that dscCfaE elicits robust HAI and anti-adhesin antibody responses in both mice and NHPs when given with adjuvant by IN vaccination, encouraging further evaluation of an ETEC adhesin-based vaccine approach.

  8. Observation of two different fractal structures in nanoparticle, protein and surfactant complexes

    SciTech Connect

    Mehan, Sumit Kumar, Sugam Aswal, V. K.

    2014-04-24

    Small angle neutron scattering has been carried out from a complex of nanoparticle, protein and surfactant. Although all the components are similarly (anionic) charged, we have observed strong interactions in their complex formation. It is characterized by the coexistence of two different mass fractal structures. The first fractal structure is originated from the protein and surfactant interaction and second from the depletion effect of first fractal structure leading the nanoparticle aggregation. The fractal structure of protein-surfactant complex represents to bead necklace structure of micelle-like clusters of surfactant formed along the unfolded protein chain. Its fractal dimension depends on the surfactant to protein ratio (r) and decreases with the increase in r. However, fractal dimension of nanoparticle aggregates in nanoparticle-protein complex is found to be independent of protein concentration and governed by the diffusion limited aggregation like morphology.

  9. Identifying subcellular localizations of mammalian protein complexes based on graph theory with a random forest algorithm.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Chen, Chao; Xie, Yun; Dai, Zong; Zou, Xiao-Yong

    2013-04-05

    In the post-genome era, one of the most important and challenging tasks is to identify the subcellular localizations of protein complexes, and further elucidate their functions in human health with applications to understand disease mechanisms, diagnosis and therapy. Although various experimental approaches have been developed and employed to identify the subcellular localizations of protein complexes, the laboratory technologies fall far behind the rapid accumulation of protein complexes. Therefore, it is highly desirable to develop a computational method to rapidly and reliably identify the subcellular localizations of protein complexes. In this study, a novel method is proposed for predicting subcellular localizations of mammalian protein complexes based on graph theory with a random forest algorithm. Protein complexes are modeled as weighted graphs containing nodes and edges, where nodes represent proteins, edges represent protein-protein interactions and weights are descriptors of protein primary structures. Some topological structure features are proposed and adopted to characterize protein complexes based on graph theory. Random forest is employed to construct a model and predict subcellular localizations of protein complexes. Accuracies on a training set by a 10-fold cross-validation test for predicting plasma membrane/membrane attached, cytoplasm and nucleus are 84.78%, 71.30%, and 82.00%, respectively. And accuracies for the independent test set are 81.31%, 69.95% and 81.00%, respectively. These high prediction accuracies exhibit the state-of-the-art performance of the current method. It is anticipated that the proposed method may become a useful high-throughput tool and plays a complementary role to the existing experimental techniques in identifying subcellular localizations of mammalian protein complexes. The source code of Matlab and the dataset can be obtained freely on request from the authors.

  10. Receptor affinity purification of a lipid-binding adhesin from Helicobacter pylori.

    PubMed Central

    Lingwood, C A; Wasfy, G; Han, H; Huesca, M

    1993-01-01

    Our previous work has shown that Helicobacter pylori specifically recognizes gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine in vitro. This binding specificity is shared by exoenzyme S from Pseudomonas aeruginosa, and monoclonal antibodies against this adhesin prevent the attachment of H. pylori to its lipid receptors. We now report the use of a novel, versatile affinity matrix to purify a 63-kDa exoenzyme S-like adhesin from H. pylori which is responsible for the lipid-binding specificity of this organism. Images PMID:8500882

  11. Detection and analysis of protein-protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes.

    PubMed

    Krause, Frank

    2006-07-01

    It is an essential and challenging task to unravel protein-protein interactions in their actual in vivo context. Native gel systems provide a separation platform allowing the analysis of protein complexes on a rather proteome-wide scale in a single experiment. This review focus on blue-native (BN)-PAGE as the most versatile and successful gel-based approach to separate soluble and membrane protein complexes of intricate protein mixtures derived from all biological sources. BN-PAGE is a charge-shift method with a running pH of 7.5 relying on the gentle binding of anionic CBB dye to all membrane and many soluble protein complexes, leading to separation of protein species essentially according to their size and superior resolution than other fractionation techniques can offer. The closely related colorless-native (CN)-PAGE, whose applicability is restricted to protein species with intrinsic negative net charge, proved to provide an especially mild separation capable of preserving weak protein-protein interactions better than BN-PAGE. The essential conditions determining the success of detecting protein-protein interactions are the sample preparations, e.g. the efficiency/mildness of the detergent solubilization of membrane protein complexes. A broad overview about the achievements of BN- and CN-PAGE studies to elucidate protein-protein interactions in organelles and prokaryotes is presented, e.g. the mitochondrial protein import machinery and oxidative phosphorylation supercomplexes. In many cases, solubilization with digitonin was demonstrated to facilitate an efficient and particularly gentle extraction of membrane protein complexes prone to dissociation by treatment with other detergents. In general, analyses of protein interactomes should be carried out by both BN- and CN-PAGE.

  12. Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

    PubMed Central

    Gutiérrez, C; Freire, R; Salas, M; Hermoso, J M

    1994-01-01

    The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex. Images PMID:8306969

  13. Toluene 4-Monooxygenase and its Complex with Effector Protein T4moD

    SciTech Connect

    Bailey, Lucas J.; Fox, Brian G.

    2012-10-16

    Toluene 4-monooxygenase (T4MO) is a multiprotein diiron enzyme complex that catalyzes the regiospecific oxidation of toluene to p-cresol. Catalytic function requires the presence of a small protein, called the effector protein. Effector protein exerts substantial control on the diiron hydroxylase catalytic cycle through protein-protein interactions. High-resolution crystal structures of the stoichometric hydroxylase and effector protein complex described here reveal how protein-protein interactions and reduction of the diiron center produce an active site configuration poised for reaction with O{sub 2}. Further information from crystal structures of mutated isoforms of the hydroxylase and a peroxo adduct is combined with catalytic results to give a fuller picture of the geometry of the enzyme-substrate complex used for the high fidelity oxidation of hydrocarbon substrates.

  14. Tuning structure of oppositely charged nanoparticle and protein complexes

    SciTech Connect

    Kumar, Sugam Aswal, V. K.; Callow, P.

    2014-04-24

    Small-angle neutron scattering (SANS) has been used to probe the structures of anionic silica nanoparticles (LS30) and cationic lyszyme protein (M.W. 14.7kD, I.P. ∼ 11.4) by tuning their interaction through the pH variation. The protein adsorption on nanoparticles is found to be increasing with pH and determined by the electrostatic attraction between two components as well as repulsion between protein molecules. We show the strong electrostatic attraction between nanoparticles and protein molecules leads to protein-mediated aggregation of nanoparticles which are characterized by fractal structures. At pH 5, the protein adsorption gives rise to nanoparticle aggregation having surface fractal morphology with close packing of nanoparticles. The surface fractals transform to open structures of mass fractal morphology at higher pH (7 and 9) on approaching isoelectric point (I.P.)

  15. Adenosine deaminase complexing protein (ADCP): a transformation sensitive protein with potentials of a cancer marker.

    PubMed

    Herbschleb-Voogt, E; Ten Kate, J; Meera Khan, P

    1983-01-01

    Several observations by independent investigators in the past have indicated that adenosine deaminase complexing protein (ADCP), present in considerable quantities in certain human tissues, was absent or decreased in the cancers originated from them. During the present study, electrophoretic analysis of adenosine deaminase (ADA) isozymes and radioimmunoassay for ADCP in the primary fibroblasts and the transformed as well as certain tumor derived cell lines have demonstrated that ADCP present in large quantities in the primary cells was absent or nearly absent in the transformed or tumor-derived cell lines. Though the mechanisms involved are not yet clear, the above observations indicate that ADCP has the potentials of a useful marker in the studies on transformed cells and cancer tissues.

  16. Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes

    PubMed Central

    Fonslow, Bryan R.; Kang, Seong A.; Gestaut, Daniel R.; Graczyk, Beth; Davis, Trisha N.; Sabatini, David M.; Yates, John R.

    2010-01-01

    Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15°C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (< 45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25°C) and eventually its individual protein components (30°C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4 – 6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions. PMID:20614870

  17. Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes.

    PubMed

    Fonslow, Bryan R; Kang, Seong A; Gestaut, Daniel R; Graczyk, Beth; Davis, Trisha N; Sabatini, David M; Yates, John R

    2010-08-01

    Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 degrees C) and eventually its individual protein components (30 degrees C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4-6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions.

  18. Unravelling the relationship between protein sequence and low-complexity regions entropies: Interactome implications.

    PubMed

    Martins, F; Gonçalves, R; Oliveira, J; Cruz-Monteagudo, M; Nieto-Villar, J M; Paz-y-Miño, C; Rebelo, I; Tejera, E

    2015-10-07

    Low-complexity regions are sub-sequences of biased composition in a protein sequence. The influence of these regions over protein evolution, specific functions and highly interactive capacities is well known. Although protein sequence entropy has been largely studied, its relationship with low-complexity regions and the subsequent effects on protein function remains unclear. In this work we propose a theoretical and empirical model integrating the sequence entropy with local complexity parameters. Our results indicate that the protein sequence entropy is related with the protein length, the entropies inside and outside the low-complexity regions as well as their number and average size. We found a small but significant increment in the sequence entropy of hubs proteins. In agreement with our theoretical model, this increment is highly dependent of the balance between the increment of protein length and average size of the low-complexity regions. Finally, our models and proteins analysis provide evidence supporting that modifications in the average size is more relevant in hubs proteins than changes in the number of low-complexity regions.

  19. Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: relationship with expression of adhesins and production of toxins.

    PubMed

    Blanco, M; Blanco, J E; Alonso, M P; Mora, A; Balsalobre, C; Muñoa, F; Juárez, A; Blanco, J

    1997-12-01

    A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesin-encoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap+ and/or sfa+ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing alpha-haemolysin (Hly+) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesin-encoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.

  20. Theory of polyelectrolyte adsorption on heterogeneously charged surfaces applied to soluble protein-polyelectrolyte complexes

    NASA Astrophysics Data System (ADS)

    de Vries, R.; Weinbreck, F.; de Kruif, C. G.

    2003-03-01

    Existing theoretical approaches to polymer adsorption on heterogeneous surfaces are applied to the problems of polyelectrolyte and polyampholyte adsorption on randomly charged surfaces. Also, analytical estimates are developed for the critical pH at which weakly charged polyelectrolytes and globular proteins start forming soluble complexes. Below a critical salt concentration, soluble complexes form "on the wrong side" of the protein isoelectric point due to the heterogeneity of the protein surface charge distribution. The analytical estimates are consistent with experimental data on soluble complexes in mixtures of gum arabic and whey protein isolate.

  1. Discovery of protein complexes with core-attachment structures from Tandem Affinity Purification (TAP) data.

    PubMed

    Wu, Min; Li, Xiao-Li; Kwoh, Chee-Keong; Ng, See-Kiong; Wong, Limsoon

    2012-09-01

    Many cellular functions involve protein complexes that are formed by multiple interacting proteins. Tandem Affinity Purification (TAP) is a popular experimental method for detecting such multi-protein interactions. However, current computational methods that predict protein complexes from TAP data require converting the co-complex relationships in TAP data into binary interactions. The resulting pairwise protein-protein interaction (PPI) network is then mined for densely connected regions that are identified as putative protein complexes. Converting the TAP data into PPI data not only introduces errors but also loses useful information about the underlying multi-protein relationships that can be exploited to detect the internal organization (i.e., core-attachment structures) of protein complexes. In this article, we propose a method called CACHET that detects protein complexes with Core-AttaCHment structures directly from bipartitETAP data. CACHET models the TAP data as a bipartite graph in which the two vertex sets are the baits and the preys, respectively. The edges between the two vertex sets represent bait-prey relationships. CACHET first focuses on detecting high-quality protein-complex cores from the bipartite graph. To minimize the effects of false positive interactions, the bait-prey relationships are indexed with reliability scores. Only non-redundant, reliable bicliques computed from the TAP bipartite graph are regarded as protein-complex cores. CACHET constructs protein complexes by including attachment proteins into the cores. We applied CACHET on large-scale TAP datasets and found that CACHET outperformed existing methods in terms of prediction accuracy (i.e., F-measure and functional homogeneity of predicted complexes). In addition, the protein complexes predicted by CACHET are equipped with core-attachment structures that provide useful biological insights into the inherent functional organization of protein complexes. Our supplementary material can

  2. Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

    PubMed

    Qureshi, M S; Sheikh, Q I; Hill, R; Brown, P E; Dickman, M J; Tzokov, S B; Rice, D W; Gjerde, D T; Hornby, D P

    2013-08-01

    The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

  3. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    NASA Astrophysics Data System (ADS)

    Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

    2014-07-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

  4. More Membranes, more Proteins: Complex Protein Import Mechanisms into Secondary Plastids

    PubMed Central

    Agrawal, Swati; Striepen, Boris

    2010-01-01

    Plastids are found across the tree of life in a tremendous diversity of life forms. Surprisingly they are not limited to photosynthetic organisms but also found in numerous predators and parasites. An important reason for the pervasiveness of plastids has been their ability to move laterally and to jump from one branch of the tree of life to the next through secondary endosymbiosis. Eukaryotic algae have entered endosymbiotic relationships with other eukaryotes on multiple independent occasions. The descendants of these endosymbiotic events now carry complex plastids, organelles that are bound by three or even four membranes. As in all endosymbiotic organelles most of the symbiont’s genes have been transferred to the host and their protein products have to be imported into the organelle. As four membranes might suggest, this is a complex process. The emerging mechanisms display a series of translocons that mirror the divergent ancestry of the membranes they cross. This review is written from a parasite biologist viewpoint and seeks to provide a brief overview of plastid evolution in particular for readers not already familiar with plant and algal biology and then focuses on recent molecular discoveries using genetically tractable Apicomplexa and diatoms. PMID:21036664

  5. An integrated map of HIV-human protein complexes that facilitate viral infection.

    PubMed

    Emig-Agius, Dorothea; Olivieri, Kevin; Pache, Lars; Shih, Hsin Ling; Pustovalova, Olga; Bessarabova, Marina; Young, John A T; Chanda, Sumit K; Ideker, Trey

    2014-01-01

    Recent proteomic and genetic studies have aimed to identify a complete network of interactions between HIV and human proteins and genes. This HIV-human interaction network provides invaluable information as to how HIV exploits the host machinery and can be used as a starting point for further functional analyses. We integrated this network with complementary datasets of protein function and interaction to nominate human protein complexes with likely roles in viral infection. Based on our approach we identified a global map of 40 HIV-human protein complexes with putative roles in HIV infection, some of which are involved in DNA replication and repair, transcription, translation, and cytoskeletal regulation. Targeted RNAi screens were used to validate several proteins and complexes for functional impact on viral infection. Thus, our HIV-human protein complex map provides a significant resource of potential HIV-host interactions for further study.

  6. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes.

    PubMed

    Mohammed, Hisham; Taylor, Christopher; Brown, Gordon D; Papachristou, Evaggelia K; Carroll, Jason S; D'Santos, Clive S

    2016-02-01

    Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) is a method that allows the study of protein complexes, in particular chromatin and transcription factor complexes, in a rapid and robust manner by mass spectrometry (MS). The method can be used in parallel with chromatin immunoprecipitation-sequencing (ChIP-seq) experiments to provide information on both the cistrome and interactome for a given protein. The method uses formaldehyde fixation to stabilize protein complexes. By using antibodies against the endogenous target, the cross-linked complex is immunoprecipitated, rigorously washed, and then digested into peptides while avoiding antibody contamination (on-bead digestion). By using this method, MS identification of the target protein and several dozen interacting proteins is possible using a 100-min LC-MS/MS run. The protocol does not require substantial proteomics expertise, and it typically takes 2-3 d from the collection of material to results.

  7. AMPK in Yeast: The SNF1 (Sucrose Non-fermenting 1) Protein Kinase Complex.

    PubMed

    Sanz, Pascual; Viana, Rosa; Garcia-Gimeno, Maria Adelaida

    2016-01-01

    In yeast, SNF1 protein kinase is the orthologue of mammalian AMPK complex. It is a trimeric complex composed of Snf1 protein kinase (orthologue of AMPKα catalytic subunit), Snf4 (orthologue of AMPKγ regulatory subunit), and a member of the Gal83/Sip1/Sip2 family of proteins (orthologues of AMPKβ subunit) that act as scaffolds and also regulate the subcellular localization of the complex. In this chapter, we review the recent literature on the characteristics of SNF1 complex subunits, the structure and regulation of the activity of the SNF1 complex, its role at the level of transcriptional regulation of relevant target genes and also at the level of posttranslational modification of targeted substrates. We also review the crosstalk of SNF1 complex activity with other key protein kinase pathways such as cAMP-PKA, TORC1, and PAS kinase.

  8. Construction of a protein-protein interaction network of Wilms' tumor and pathway prediction of molecular complexes.

    PubMed

    Teng, W J; Zhou, C; Liu, L J; Cao, X J; Zhuang, J; Liu, G X; Sun, C G

    2016-05-23

    Wilms' tumor (WT), or nephroblastoma, is the most common malignant renal cancer that affects the pediatric population. Great progress has been achieved in the treatment of WT, but it cannot be cured at present. Nonetheless, a protein-protein interaction network of WT should provide some new ideas and methods. The purpose of this study was to analyze the protein-protein interaction network of WT. We screened the confirmed disease-related genes using the Online Mendelian Inheritance in Man database, created a protein-protein interaction network based on biological function in the Cytoscape software, and detected molecular complexes and relevant pathways that may be included in the network. The results showed that the protein-protein interaction network of WT contains 654 nodes, 1544 edges, and 5 molecular complexes. Among them, complex 1 is predicted to be related to the Jak-STAT signaling pathway, regulation of hematopoiesis by cytokines, cytokine-cytokine receptor interaction, cytokine and inflammatory responses, and hematopoietic cell lineage pathways. Molecular complex 4 shows a correlation of WT with colorectal cancer and the ErbB signaling pathway. The proposed method can provide the bioinformatic foundation for further elucidation of the mechanisms of WT development.

  9. Glycosaminoglycan binding by Borrelia burgdorferi adhesin BBK32 specifically and uniquely promotes joint colonization

    PubMed Central

    Lin, Yi-Pin; Chen, Qiang; Ritchie, Jennifer A.; Dufour, Nicholas P.; Fischer, Joshua R.; Coburn, Jenifer; Leong, John M.

    2014-01-01

    SUMMARY Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins that mediate attachment to cells and/or extracellular matrix in target organs. Many of these ‘adhesins’ bind to multiple ligands, complicating efforts to understand the role of each ligand-binding activity. Borrelia burgdorferi, the causative agent of Lyme disease, produces BBK32, first identified as a fibronectin-binding adhesin that promotes skin and joint colonization. BBK32 also binds to glycosaminoglycan (GAG), which, like fibronectin is ubiquitously present on cell surfaces. To determine which binding activity is relevant for BBK32-promoted infectivity, we generated a panel of BBK32 truncation and internal deletion mutants, and identified variants specifically defective for binding to either fibronectin or GAG. These variants promoted bacterial attachment to different mammalian cell types in vitro, suggesting that fibronectin and GAG binding may play distinct roles during infection. Intravenous inoculation of mice with a high-passage non-infectious B. burgdorferi strain that produced wild type BBK32 or BBK32 mutants defective for GAG or fibronectin binding, revealed that only GAG-binding activity was required for significant localization to joints at 60 minutes post-infection. An otherwise infectious B. burgdorferi strain producing BBK32 specifically deficient in fibronectin binding was fully capable of both skin and joint colonization in the murine model, whereas a strain producing BBK32 selectively attenuated for GAG binding colonized the inoculation site but not knee or tibiotarsus joints. Thus, the BBK32 fibronectin- and GAG-binding activities are separable in vivo, and BBK32-mediated GAG binding, but not fibronectin binding, contributes to joint colonization. PMID:25486989

  10. New vehicles allow detergent-free mass spectrometry of membrane protein complexes

    PubMed Central

    Hopper, Jonathan T.S.; Yu, Yvonne Ting-Chun; Li, Dianfan; Raymond, Alison; Bostock, Mark; Liko, Idlir; Mikhailov, Victor; Laganowsky, Arthur; Benesch, Justin L.P.; Caffrey, Martin; Nietlispach, Daniel; Robinson, Carol V.

    2013-01-01

    The rapid growth in the study of membrane protein complexes in lipid microenvironments prompts new methods for their characterization. Here we develop mass spectrometry as a method for studying membrane proteins incorporated into amphipols, bicelles and nanodiscs. We compare these detergent-free vehicles with micelles for their ability to preserve the interactions of oligomeric complexes and to stabilize proteins that require defined lipid environments. PMID:24122040

  11. Structural Basis for the Coevolution of a Viral RNA-Protein Complex

    SciTech Connect

    Chao,J.; Patskovsky, Y.; Almo, S.; Singer, R.

    2008-01-01

    The cocrystal structure of the PP7 bacteriophage coat protein in complex with its translational operator identifies a distinct mode of sequence-specific RNA recognition when compared to the well-characterized MS2 coat protein-RNA complex. The structure reveals the molecular basis of the PP7 coat protein's ability to selectively bind its cognate RNA, and it demonstrates that the conserved beta-sheet surface is a flexible architecture that can evolve to recognize diverse RNA hairpins.

  12. Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling.

    PubMed

    Pertl-Obermeyer, Heidi; Wu, Xu Na; Schrodt, Jens; Müdsam, Christina; Obermeyer, Gerhard; Schulze, Waltraud X

    2016-09-01

    Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3β and ap-4β knock-out mutants. In ap-3β mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4β mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3β and ap-4β compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking.

  13. An informatic framework for decoding protein complexes by top-down mass spectrometry.

    PubMed

    Skinner, Owen S; Havugimana, Pierre C; Haverland, Nicole A; Fornelli, Luca; Early, Bryan P; Greer, Joseph B; Fellers, Ryan T; Durbin, Kenneth R; Do Vale, Luis H F; Melani, Rafael D; Seckler, Henrique S; Nelp, Micah T; Belov, Mikhail E; Horning, Stevan R; Makarov, Alexander A; LeDuc, Richard D; Bandarian, Vahe; Compton, Philip D; Kelleher, Neil L

    2016-03-01

    Efforts to map the human protein interactome have resulted in information about thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we report a computational search strategy that supports hierarchical top-down analysis for precise identification and scoring of multi-proteoform complexes by native mass spectrometry.

  14. A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

    PubMed Central

    2011-01-01

    Background Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology. Results A two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation. Conclusion Two dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes. PMID:21269440

  15. Determining Protein Complex Connectivity Using a Probabilistic Deletion Network Derived from Quantitative Proteomics

    PubMed Central

    Sardiu, Mihaela E.; Gilmore, Joshua M.; Carrozza, Michael J.; Li, Bing; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.

    2009-01-01

    Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex. PMID:19806189

  16. Cooperative Role for Tetraspanins in Adhesin-Mediated Attachment of Bacterial Species to Human Epithelial Cells ▿ †

    PubMed Central

    Green, Luke R.; Monk, Peter N.; Partridge, Lynda J.; Morris, Paul; Gorringe, Andrew R.; Read, Robert C.

    2011-01-01

    The tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins. Neisseria meningitidis, the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function in Neisseria meningitidis adherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence of Neisseria meningitidis. In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells of Neisseria meningitidis strain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains of Staphylococcus aureus, Neisseria lactamica, Escherichia coli, and Streptococcus pneumoniae was also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors for Neisseria meningitidis and potentially for multiple species of bacteria. PMID:21464080

  17. The HIR corepressor complex binds to nucleosomes generating a distinct protein/DNA complex resistant to remodeling by SWI/SNF

    PubMed Central

    Prochasson, Philippe; Florens, Laurence; Swanson, Selene K.; Washburn, Michael P.; Workman, Jerry L.

    2005-01-01

    The histone regulatory (HIR) and histone promoter control (HPC) repressor proteins regulate three of the four histone gene loci during the Saccharomyces cerevisiae cell cycle. Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 proteins form a stable HIR repressor complex. The HIR complex promotes histone deposition onto DNA in vitro and constitutes a novel nucleosome assembly complex. The HIR complex stably binds to DNA and nucleosomes. Furthermore, HIR complex binding to nucleosomes forms a distinct protein/DNA complex resistant to remodeling by SWI/SNF. Thus, the HIR complex is a novel nucleosome assembly complex which functions with SWI/SNF to regulate transcription. PMID:16264190

  18. A single Sec61-complex functions as a protein-conducting channel.

    PubMed

    Kalies, Kai-Uwe; Stokes, Vivica; Hartmann, Enno

    2008-12-01

    During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61alpha-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.

  19. Understanding the nanoparticle-protein corona complexes using computational and experimental methods.

    PubMed

    Kharazian, B; Hadipour, N L; Ejtehadi, M R

    2016-06-01

    Nanoparticles (NP) have capability to adsorb proteins from biological fluids and form protein layer, which is called protein corona. As the cell sees corona coated NPs, the protein corona can dictate biological response to NPs. The composition of protein corona is varied by physicochemical properties of NPs including size, shape, surface chemistry. Processing of protein adsorption is dynamic phenomena; to that end, a protein may desorb or leave a surface vacancy that is rapidly filled by another protein and cause changes in the corona composition mainly by the Vroman effect. In this review, we discuss the interaction between NP and proteins and the available techniques for identification of NP-bound proteins. Also we review current developed computational methods for understanding the NP-protein complex interactions.

  20. Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

    NASA Technical Reports Server (NTRS)

    Cannone, Jaime J.; Barnes, Cindy L.; Achari, Aniruddha; Kundrot, Craig E.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

  1. Folding and self-assembly of a small protein complex

    PubMed Central

    Sieradzan, Adam K.; Liwo, Adam; Hansmann, Ulrich H.E.

    2012-01-01

    The synthetic homotetrameric ββα (BBAT1) protein possesses a stable quaternary structure with a ββα fold. Because of its small size (a total of 84 residues), the homotetramer is an excellent model system with which to study the self-assembly and protein-protein interactions. We find from replica exchange molecular dynamics simulations with the coarse-grain UNRES force field that the folding and association pathway consists of three well-separated steps, where that association to a tetramer precedes and facilitates folding of the four chains. At room temperature the tetramer exists in an ensemble of diverse structures. The crystal structure becomes energetically favored only when the molecule is put in a dense and crystal-like environment. The observed picture of folding promoted by association may mirror the mechanism according to which intrinsically unfolded proteins assume their functional structure. PMID:24039552

  2. Protein trafficking to the complex chloroplasts of Euglena.

    PubMed

    Vacula, Rostislav; Sláviková, Silvia; Schwartzbach, Steven D

    2007-01-01

    Proteins are delivered to Euglena chloroplasts using the secretory pathway. We describe analytical methods to study the intracellular trafficking of Euglena chloroplast proteins and a method to isolate preparative amounts of intact import competent chloroplasts for biochemical studies. Cells are pulse labeled with 35S-sulfate and chased with unlabeled sulfate allowing the trafficking and posttranslational processing of the labeled protein to be followed. Sucrose gradients are used to separate a 35S-labeled cell lysate into cytoplasmic, endoplasmic reticuum (ER), Golgi apparatus, chloroplast and mitochondrial fractions. Immunoprecipitation of each gradient fraction allows identification of the intracellular compartment containing a specific 35S-labeled protein at different times after synthesis delineating the trafficking pathway. Because sucrose gradients cannot be used to isolate preparative amounts of highly purified chloroplasts for biochemical characterization, a preparative high-yield procedure using Percoll gradients to isolate highly purified import competent chloroplasts is also presented.

  3. Structural insights into yeast histone chaperone Hif1: a scaffold protein recruiting protein complexes to core histones.

    PubMed

    Liu, Hejun; Zhang, Mengying; He, Wei; Zhu, Zhongliang; Teng, Maikun; Gao, Yongxiang; Niu, Liwen

    2014-09-15

    Yeast Hif1 [Hat1 (histone acetyltransferase 1)-interacting factor], a homologue of human NASP (nuclear autoantigenic sperm protein), is a histone chaperone that is involved in various protein complexes which modify histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, in the present paper we demonstrate the crystal structure of Hif1 consisting of a superhelixed TPR (tetratricopeptide repeat) domain and an extended acid loop covering the rear of TPR domain, which represent typical characteristics of SHNi-TPR [Sim3 (start independent of mitosis 3)-Hif1-NASP interrupted TPR] proteins. Our binding assay indicates that Hif1 could bind to the histone octamer via histones H3 and H4. The acid loop is shown to be crucial for the binding of histones and may also change the conformation of the TPR groove. By binding to the core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly.

  4. Identification and Validation of PTEN Complex, Associated Proteins

    DTIC Science & Technology

    2005-11-01

    Rosalia construct was transcribed and translated using a wheat germ lysate transcription/translation system to generate an unphosphorylated protein...efficient using the wheat germ lysate transcription/translation, system the new antisera immunoprecipitated the protein as well as the C54 Ab, especially...pSGL-PTEN was in vitro translated in a Rabbit reticolocyte lysate system (A) or in a wheat germ system (B) in the presence of radioactively labeled

  5. Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1:1 syntaxin/SNAP-25 complex

    PubMed Central

    Weninger, Keith; Bowen, Mark E.; Choi, Ucheor B.; Chu, Steven; Brunger, Axel T.

    2010-01-01

    Summary Formation of a binary complex between syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) at the active zone is believed to precede assembly of the ternary SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex that is essential for neurotransmitter release. Despite its importance in models of synaptic neurotransmitter release, this binary complex has been difficult to characterize by bulk methods due to the prevalence of a 2:1 dead-end species. Here we used single molecule fluorescence resonance energy transfer (smFRET) to study the structure and dynamics of the 1:1 syntaxin/SNAP-25 binary complex. The binary complex is conformationally variable with FRET efficiency states often changing on the second timescale. One state corresponds to a parallel three-helix bundle configuration, while other states correspond to configurations with one of the SNAP-25 SNARE domains dissociated. All configurations of the binary complex are rapidly locked into the single three-helix bundle configuration by the addition of synaptobrevin. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex serves as a dynamic acceptor for synaptobrevin binding, and interactions with accessory proteins stabilize this acceptor. In a high protein density cellular environment the syntaxin/SNAP-25 complex is therefore expected to be in the configuration where it can rapidly interact with synaptobrevin so its formation is unlikely a limiting step for SNARE-mediated neurotransmitter release. PMID:18275821

  6. Kojak: Efficient analysis of chemically cross-linked protein complexes

    PubMed Central

    Hoopmann, Michael R.; Zelter, Alex; Johnson, Richard S.; Riffle, Michael; MacCoss, Michael J.; Davis, Trisha N.; Moritz, Robert L.

    2015-01-01

    Protein chemical cross-linking and mass spectrometry enable the analysis of protein-protein interactions and protein topologies, however complicated cross-linked peptide spectra require specialized algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein-protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms, and can identify cross-linked peptides using many different chemical cross-linkers, with or without heavy isotope labels. Kojak was used to analyze both novel and existing datasets, and was compared with existing cross-linking algorithms. The algorithm provided increased cross-link identifications over existing algorithms, and equally importantly, the results in a fraction of computational time. The Kojak algorithm is open-source, cross-platform, and freely available. This software provides both existing and new cross-linking researchers alike an effective way to derive additional cross-link identifications from new or existing datasets. For new users, it provides a simple analytical resource resulting in more cross-link identifications than other methods. PMID:25812159

  7. Identification of a preassembled TRH receptor-G(q/11) protein complex in HEK293 cells.

    PubMed

    Drastichova, Zdenka; Novotny, Jiri

    2012-01-01

    Protein-protein interactions define specificity in signal transduction and these interactions are central to transmembrane signaling by G-protein-coupled receptors (GPCRs). It is not quite clear, however, whether GPCRs and the regulatory trimeric G-proteins behave as freely and independently diffusible molecules in the plasma membrane or whether they form some preassociated complexes. Here we used clear-native polyacrylamide gel electrophoresis (CN-PAGE) to investigate the presumed coupling between thyrotropin-releasing hormone (TRH) receptor and its cognate G(q/11) protein in HEK293 cells expressing high levels of these proteins. Under different solubilization conditions, the TRH receptor (TRH-R) was identified to form a putative pentameric complex composed of TRH-R homodimer and G(q/11) protein. The presumed association of TRH-R with G(q/11)α or Gβ proteins in plasma membranes was verified by RNAi experiments. After 10- or 30-min hormone treatment, TRH-R signaling complexes gradually dissociated with a concomitant release of receptor homodimers. These observations support the model in which GPCRs can be coupled to trimeric G-proteins in preassembled signaling complexes, which might be dynamically regulated upon receptor activation. The precoupling of receptors with their cognate G-proteins can contribute to faster G-protein activation and subsequent signal transfer into the cell interior.

  8. Comparative evolutionary analysis of protein complexes in E. coli and yeast

    PubMed Central

    2010-01-01

    Background Proteins do not act in isolation; they frequently act together in protein complexes to carry out concerted cellular functions. The evolution of complexes is poorly understood, especially in organisms other than yeast, where little experimental data has been available. Results We generated accurate, high coverage datasets of protein complexes for E. coli and yeast in order to study differences in the evolution of complexes between these two species. We show that substantial differences exist in how complexes have evolved between these organisms. A previously proposed model of complex evolution identified complexes with cores of interacting homologues. We support findings of the relative importance of this mode of evolution in yeast, but find that it is much less common in E. coli. Additionally it is shown that those homologues which do cluster in complexes are involved in eukaryote-specific functions. Furthermore we identify correlated pairs of non-homologous domains which occur in multiple protein complexes. These were identified in both yeast and E. coli and we present evidence that these too may represent complex cores in yeast but not those of E. coli. Conclusions Our results suggest that there are differences in the way protein complexes have evolved in E. coli and yeast. Whereas some yeast complexes have evolved by recruiting paralogues, this is not apparent in E. coli. Furthermore, such complexes are involved in eukaryotic-specific functions. This implies that the increase in gene family sizes seen in eukaryotes in part reflects multiple family members being used within complexes. However, in general, in both E. coli and yeast, homologous domains are used in different complexes. PMID:20122144

  9. Fast Photochemical Oxidation of Proteins (FPOP) for Comparing Structures of Protein/Ligand Complexes: The Calmodulin-peptide Model System

    PubMed Central

    Zhang, Hao; Gau, Brian C.; Jones, Lisa M.; Vidavsky, Ilan; Gross, Michael L.

    2010-01-01

    Fast Photochemical Oxidation of Proteins (FPOP) is a mass-spectrometry-based protein footprinting method that modifies proteins on the microsecond time scale. Highly reactive •OH, produced by laser photolysis of hydrogen peroxide, oxidatively modifies the side chains of approximately one half the common amino acids on this time scale. Owing to the short labeling exposure, only solvent-accessible residues are sampled. Quantification of the modification extent for the apo and holo states of a protein-ligand complex should provide structurally sensitive information at the amino-acid level to compare the structures of unknown protein complexes with known ones. We report here the use of FPOP to monitor the structural changes of calmodulin in its established binding to M13 of the skeletal muscle myosin light chain kinase. We use the outcome to establish the unknown structures resulting from binding with melittin and mastoparan. The structural comparison follows from a comprehensive examination of the extent of FPOP modifications as measured by proteolysis and LC-MS/MS for each protein-ligand equilibrium. The results not only show that the three calmodulin-peptide complexes have similar structures but also reveal those regions of the protein that became more or less solvent-accessible upon binding. This approach has the potential for relatively high throughput, information-dense characterization of a series of protein-ligand complexes in biochemistry and drug discovery when the structure of one reference complex is known, as is the case for calmodulin and M13 of the skeletal muscle myosin light chain kinase, and the structures of related complexes are not,. PMID:21142124

  10. A Staphylococcus aureus Proteome Overview: Shared and Specific Proteins and Protein Complexes from Representative Strains of All Three Clades.

    PubMed

    Liang, Chunguang; Schaack, Dominik; Srivastava, Mugdha; Gupta, Shishir K; Sarukhanyan, Edita; Giese, Anne; Pagels, Martin; Romanov, Natalie; Pané-Farré, Jan; Fuchs, Stephan; Dandekar, Thomas

    2016-02-19

    Staphylococcus aureus is an important model organism and pathogen. This S. aureus proteome overview details shared and specific proteins and selected virulence-relevant protein complexes from representative strains of all three major clades. To determine the strain distribution and major clades we used a refined strain comparison combining ribosomal RNA, MLST markers, and looking at highly-conserved regions shared between strains. This analysis shows three sub-clades (A-C) for S. aureus. As calculations are complex and strain annotation is quite time consuming we compare here key representatives of each clade with each other: model strains COL, USA300, Newman, and HG001 (clade A), model strain N315 and Mu50 (clade B) and ED133 and MRSA252 (clade C). We look at these individual proteomes and compare them to a background of 64 S. aureus strains. There are overall 13,284 S. aureus proteins not part of the core proteome which are involved in different strain-specific or more general complexes requiring detailed annotation and new experimental data to be accurately delineated. By comparison of the eight representative strains, we identify strain-specific proteins (e.g., 18 in COL, 105 in N315 and 44 in Newman) that characterize each strain and analyze pathogenicity islands if they contain such strain-specific proteins. We identify strain-specific protein repertoires involved in virulence, in cell wall metabolism, and phosphorylation. Finally we compare and analyze protein complexes conserved and well-characterized among S. aureus (a total of 103 complexes), as well as predict and analyze several individual protein complexes, including structure modeling in the three clades.

  11. A Staphylococcus aureus Proteome Overview: Shared and Specific Proteins and Protein Complexes from Representative Strains of All Three Clades

    PubMed Central

    Liang, Chunguang; Schaack, Dominik; Srivastava, Mugdha; Gupta, Shishir K.; Sarukhanyan, Edita; Giese, Anne; Pagels, Martin; Romanov, Natalie; Pané-Farré, Jan; Fuchs, Stephan; Dandekar, Thomas

    2016-01-01

    Staphylococcus aureus is an important model organism and pathogen. This S. aureus proteome overview details shared and specific proteins and selected virulence-relevant protein complexes from representative strains of all three major clades. To determine the strain distribution and major clades we used a refined strain comparison combining ribosomal RNA, MLST markers, and looking at highly-conserved regions shared between strains. This analysis shows three sub-clades (A–C) for S. aureus. As calculations are complex and strain annotation is quite time consuming we compare here key representatives of each clade with each other: model strains COL, USA300, Newman, and HG001 (clade A), model strain N315 and Mu50 (clade B) and ED133 and MRSA252 (clade C). We look at these individual proteomes and compare them to a background of 64 S. aureus strains. There are overall 13,284 S. aureus proteins not part of the core proteome which are involved in different strain-specific or more general complexes requiring detailed annotation and new experimental data to be accurately delineated. By comparison of the eight representative strains, we identify strain-specific proteins (e.g., 18 in COL, 105 in N315 and 44 in Newman) that characterize each strain and analyze pathogenicity islands if they contain such strain-specific proteins. We identify strain-specific protein repertoires involved in virulence, in cell wall metabolism, and phosphorylation. Finally we compare and analyze protein complexes conserved and well-characterized among S. aureus (a total of 103 complexes), as well as predict and analyze several individual protein complexes, including structure modeling in the three clades. PMID:28248218

  12. Identification of a novel protein complex containing annexin A4, rabphilin and synaptotagmin.

    PubMed

    Willshaw, Angela; Grant, Karen; Yan, Jun; Rockliffe, Nichola; Ambavarapu, Sailaja; Burdyga, Galina; Varro, Andrea; Fukuoka, Shin-Ichi; Gawler, Debra

    2004-02-13

    Rabphilin is a synaptic vesicle-associated protein proposed to play a role in regulating neurotransmitter release. Here we report the isolation and identification of a novel protein complex containing rabphilin, annexin A4 and synaptotagmin 1. We show that the rabphilin C2B domain interacts directly with the N-terminus of annexin A4 and mediates the co-complexing of these two proteins in PC12 cells. Analyzing the cellular localisation of these co-complexing proteins we find that annexin A4 is located on synaptic membranes and co-localises with rabphilin at the plasma membrane in PC12 cells. Given that rabphilin and synaptotagmin are synaptic vesicle proteins involved in neurotransmitter release, the identification of this complex suggests that annexin A4 may play a role in synaptic exocytosis.

  13. Monte Carlo simulations of flexible polyanions complexing with whey proteins at their isoelectric point

    NASA Astrophysics Data System (ADS)

    de Vries, R.

    2004-02-01

    Electrostatic complexation of flexible polyanions with the whey proteins α-lactalbumin and β-lactoglobulin is studied using Monte Carlo simulations. The proteins are considered at their respective isoelectric points. Discrete charges on the model polyelectrolytes and proteins interact through Debye-Hückel potentials. Protein excluded volume is taken into account through a coarse-grained model of the protein shape. Consistent with experimental results, it is found that α-lactalbumin complexes much more strongly than β-lactoglobulin. For α-lactalbumin, strong complexation is due to localized binding to a single large positive "charge patch," whereas for β-lactoglobulin, weak complexation is due to diffuse binding to multiple smaller charge patches.

  14. Human protein HC and its IgA complex are inhibitors of neutrophil chemotaxis.

    PubMed Central

    Méndez, E; Fernández-Luna, J L; Grubb, A; Leyva-Cobián, F

    1986-01-01

    Protein HC, a heterogeneously charged low molecular weight glycoprotein, and its IgA complex were isolated from human plasma and urine. Plasma from individuals with monoclonal IgA populations was used as starting material for the isolation of the protein HC-IgA complex to obtain homogeneous complex populations. Neither low molecular weight protein HC nor its IgA complex in the concentrations 30 and 600 mg/liter influenced the random migration of normal human neutrophils. The chemotactic response of neutrophils to endotoxin-activated serum was, however, attenuated in a dose-dependent way by both low molecular weight protein HC and protein HC-IgA complex. Concentrations of protein HC and its IgA complex producing significant inhibition of the chemotactic response were found to occur in plasma from healthy and diseased individuals as well as in synovial fluid from patients with rheumatoid arthritis. These results suggest that protein HC and its IgA complex play physiological roles in the regulation of the inflammatory response. PMID:2419908

  15. SVP-like MADS-box protein from Carya cathayensis forms higher-order complexes.

    PubMed

    Wang, Jingjing; Hou, Chuanming; Huang, Jianqin; Wang, Zhengjia; Xu, Yingwu

    2015-03-01

    To properly regulate plant flowering time and construct floral pattern, MADS-domain containing transcription factors must form multimers including homo- and hetero-dimers. They are also active in forming hetero-higher-order complexes with three to five different molecules. However, it is not well known if a MADS-box protein can also form homo-higher-order complex. In this study a biochemical approach is utilized to provide insight into the complex formation for an SVP-like MADS-box protein cloned from hickory. The results indicated that the protein is a heterogeneous higher-order complex with the peak population containing over 20 monomers. Y2H verified the protein to form homo-complex in yeast cells. Western blot of the hickory floral bud sample revealed that the protein exists in higher-order polymers in native. Deletion assays indicated that the flexible C-terminal residues are mainly responsible for the higher-order polymer formation and the heterogeneity. Current results provide direct biochemical evidences for an active MADS-box protein to be a high order complex, much higher than a quartermeric polymer. Analysis suggests that a MADS-box subset may be able to self-assemble into large complexes, and thereby differentiate one subfamily from the other in a higher-order structural manner. Present result is a valuable supplement to the action of mechanism for MADS-box proteins in plant development.

  16. Purification of RNA-Protein Splicing Complexes Using a Tagged Protein from In Vitro Splicing Reaction Mixture.

    PubMed

    Kataoka, Naoyuki

    2016-01-01

    In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.

  17. Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map.

    PubMed

    Collins, Sean R; Miller, Kyle M; Maas, Nancy L; Roguev, Assen; Fillingham, Jeffrey; Chu, Clement S; Schuldiner, Maya; Gebbia, Marinella; Recht, Judith; Shales, Michael; Ding, Huiming; Xu, Hong; Han, Junhong; Ingvarsdottir, Kristin; Cheng, Benjamin; Andrews, Brenda; Boone, Charles; Berger, Shelley L; Hieter, Phil; Zhang, Zhiguo; Brown, Grant W; Ingles, C James; Emili, Andrew; Allis, C David; Toczyski, David P; Weissman, Jonathan S; Greenblatt, Jack F; Krogan, Nevan J

    2007-04-12

    Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal; however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.

  18. A maize protein associated with the G-box binding complex has homology to brain regulatory proteins.

    PubMed Central

    de Vetten, N C; Lu, G; Feri, R J

    1992-01-01

    The G-box element is a moderately conserved component of the promoter of many inducible genes, including the alcohol dehydrogenase genes of Arabidopsis and maize. We used monoclonal antibodies generated against partially purified G-box binding factor (GBF) activity to characterize maize proteins that are part of the DNA binding complex. Antibodies interacted with partially purified maize GBF complexes to produce a slower migrating complex in the gel retardation assay. Immunoprecipitation experiments suggested that the protein recognized by the antibody is not a DNA binding protein in and of itself, but rather is associated with the DNA binding complex. These monoclonal antibodies were used to isolate cDNA clones encoding a protein that we have designated GF14. Maize GF14 contains a region resembling a leucine zipper and acidic carboxy and amino termini, of which the latter can form an amphipathic alpha-helix similar to known transcriptional activators such as VP16 and GAL4. Protein gel blot analysis of cell culture extract showed that a single, major protein of approximately 30 kD is recognized by anti-GF14; the protein is also present predominantly in the kernel and root. The deduced amino acid sequence of maize GF14 is more than 80% identical to Arabidopsis GF14 and Oenothera PHP-O, and is more than 60% identical to a class of mammalian brain proteins described as both protein kinase C inhibitors and activators of tyrosine and tryptophan hydroxylases. GF14 is found in a variety of monocotyledons and dicotyledons, gymnosperms, and yeast. This suggests a deep evolutionary conservation of a potential regulatory protein associated with a core sequence found in the promoter region of many genes. PMID:1446170

  19. Complexation-triggerable liposome mixed with silk protein and chitosan.

    PubMed

    Hong, Yeon-Ji; Kim, Jin-Chul

    2015-01-01

    Complexation-triggerable liposomes were prepared by modifying the surface of egg phosphatidylcholine (EPC) liposomes with hydrophobicized silk fibroin (HmSF) and hydrophobicized chitosan (HmCh). Maximum complexation, determined by measuring the diameter of complexation, was found when the ratio of HmSF to HmCh was 14:1, so they were immobilized on the surface of liposomes at the same ratio. The degree of fluorescence quenching of calcein in liposomal suspension was as high as 68% when the ratio of surface modifier (HmSF + HmCh) to EPC was 1:15. When the ratio was increased to 1:5, the degree of quenching decreased to 32%, indicating the inefficient formation of liposome. Liposome mixed with the surface modifier was multi-lamellar vesicle on TEM photo. And, the mean diameter was larger than those of liposome mixed with either HmSF or HmCh, possibly due to insoluble complex on the liposomal surface. The liposome exhibited a pH-sensitive release and triggered the release at pH 5.5 and 6.0. It is believed that complexation is responsible for the promoted release at those pH values.

  20. Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation

    PubMed Central

    Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd

    2016-01-01

    Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380

  1. Dynamic Complexes in the Chaperonin-Mediated Protein Folding Cycle

    PubMed Central

    Weiss, Celeste; Jebara, Fady; Nisemblat, Shahar; Azem, Abdussalam

    2016-01-01

    The GroEL–GroES chaperonin system is probably one of the most studied chaperone systems at the level of the molecular mechanism. Since the first reports of a bacterial gene involved in phage morphogenesis in 1972, these proteins have stimulated intensive research for over 40 years. During this time, detailed structural and functional studies have yielded constantly evolving concepts of the chaperonin mechanism of action. Despite of almost three decades of research on this oligomeric protein, certain aspects of its function remain controversial. In this review, we highlight one central aspect of its function, namely, the active intermediates of its reaction cycle, and present how research to this day continues to change our understanding of chaperonin-mediated protein folding. PMID:28008398

  2. Leptospira surface adhesin (Lsa21) induces Toll like receptor 2 and 4 mediated inflammatory responses in macrophages

    PubMed Central

    Faisal, Syed M.; Varma, Vivek P.; Subathra, M.; Azam, Sarwar; Sunkara, Anil K.; Akif, Mohd; Baig, Mirza. S.; Chang, Yung-Fu

    2016-01-01

    Leptospirosis is zoonotic and emerging infectious disease of global importance. Little is understood about Leptospira pathogenesis and host immune response. In the present work we have investigated how Leptospira modulates the host innate immune response mediated by Toll-like receptors (TLRs) via surface exposed proteins. We screened Leptospira outer membrane/surface proteins for their ability to activate/inhibit TLR2/4 signaling in HEK293 cell lines. Of these the 21 kDa Leptospira surface adhesin, Lsa21 had strong TLR2 and TLR4 activity leading to production of proinflammatory cytokines and expression of costimulatory molecules in mouse macrophages. This activity of Lsa21 on innate response was dependent on activation of mitogen activated protein kinases (MAPKs) via stimulating the rapid phosphorylation of p38, JNK and activation of transcription factor NF-κB. Additionally, neutralizing antibodies against TLR2 and TLR4 significantly inhibited cytokine secretion and attenuated Lsa21 induced phosphorylation of p38 and JNK. Furthermore, Lsa21 induced cytokine levels were significantly lower in TLR2−/− and TLR4−/− than in wild type mouse macrophage cell lines. Confocal microscopy and molecular docking confirmed that Lsa21 interacted with both TLR2 and TLR4. These results indicate that Lsa21 is a potent TLR2 and TLR4 agonist that induces strong innate response and may play important role in Leptospira pathogenesis. PMID:27996041

  3. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

    SciTech Connect

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion

    2010-08-18

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{sub 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

  4. Protein Crystal Eco R1 Endonulease-DNA Complex

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Type II restriction enzymes, such as Eco R1 endonulease, present a unique advantage for the study of sequence-specific recognition because they leave a record of where they have been in the form of the cleaved ends of the DNA sites where they were bound. The differential behavior of a sequence -specific protein at sites of differing base sequence is the essence of the sequence-specificity; the core question is how do these proteins discriminate between different DNA sequences especially when the two sequences are very similar. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  5. Pigment-Protein Complexes of Illuminated Etiolated Leaves

    PubMed Central

    Oliver, Richard P.; Griffiths, W. Trevor

    1982-01-01

    Photoconversion of protochlorophyllide in etiolated leaves of Avena sativa L., var. Pennal or Peniarth and Phaseolus vulgare L., var. `The Prince' results in the sequential appearance of spectrally distinct chlorophyllide complexes (Chlide 678, 684, and 672). This paper reports on the generation of similar forms in vitro, under controlled conditions, using well characterized etioplast membranes enriched in the enzyme protochlorophyllide reductase. Excess NADP+ and NADPH stabilize complexes related to Chlide 678 and Chlide 684, respectively, whereas addition of exogenous Pchlide induces formation of a species related to Chlide 672. Evidence is provided to support the suggestion that Chlide 678 and Chlide 684 represent ternary complexes of the enzyme protochlorophyllide reductase, with Chlide and either NADP+ (Chlide 678) or NADPH (Chlide 684). Chlide 672 is seen as `free' pigment dissociated from the enzyme. The role of Pchlide in this dissociation, observed spectroscopically as the `Shibata shift,' is discussed. PMID:16662606

  6. Modifications in structure and interaction of nanoparticle-protein-surfactant complexes in electrolyte solution

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Kumar, S.; Aswal, V. K.; Schweins, R.

    2016-05-01

    SANS experiments of three-component system of anionic silica nanoparticles, anionic BSA protein and anionic SDS surfactants have been carried out without and with electrolyte in aqueous solution. In both the cases, the interaction of surfactant with protein results in formation of bead-necklace structure of protein-surfactant complexes in solution. These protein-surfactant complexes interact very differently with nanoparticles in absence and presence of electrolyte. In absence of electrolyte, nanoparticles remain in dispersed phase in solution, whereas with the addition of electrolyte the nanoparticles fractal aggregates are formed. SANS describes the phase behavior to be governed by competition of electrostatic and depletion interactions among the components solution.

  7. A Review of Methods Used for Identifying Structural Changes in a Large Protein Complex

    PubMed Central

    Nadeau, Owen W.; Carlson, Gerald M.

    2013-01-01

    This chapter explores the structural responses of a massive, hetero-oligomeric protein complex to a single allosteric activator as probed by a wide range of chemical, biochemical, and biophysical approaches. Some of the approaches used are amenable only to large protein targets, whereas others push the limits of their utility. Some of the techniques focus on individual subunits, or portions thereof, while others examine the complex as a whole. Despite the absence of crystallographic data for the complex, the diverse techniques identify and implicate a small region of its catalytic subunit as the master allosteric activation switch for the entire complex. PMID:22052488

  8. Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP.

    PubMed

    Maita, Nobuo; Okada, Kengo; Hatakeyama, Kazuyuki; Hakoshima, Toshio

    2002-02-05

    In the presence of phenylalanine, GTP cyclohydrolase I feedback regulatory protein (GFRP) forms a stimulatory 360-kDa complex with GTP cyclohydrolase I (GTPCHI), which is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. The crystal structure of the stimulatory complex reveals that the GTPCHI decamer is sandwiched by two GFRP homopentamers. Each GFRP pentamer forms a symmetrical five-membered ring similar to beta-propeller. Five phenylalanine molecules are buried inside each interface between GFRP and GTPCHI, thus enhancing the binding of these proteins. The complex structure suggests that phenylalanine-induced GTPCHI x GFRP complex formation enhances GTPCHI activity by locking the enzyme in the active state.

  9. The topology and dynamics of protein complexes: insights from intra- molecular network theory.

    PubMed

    Hu, Guang; Zhou, Jianhong; Yan, Wenying; Chen, Jiajia; Shen, Bairong

    2013-03-01

    Intra-molecular interactions within complex systems play a pivotal role in the biological function. They form a major challenge to computational structural proteomics. The network paradigm treats any system as a set of nodes linked by edges corresponding to the relations existing between the nodes. It offers a computationally efficient tool to meet this challenge. Here, we review the recent advances in the use of network theory to study the topology and dynamics of protein- ligand and protein-nucleic acid complexes. The study of protein complexes networks not only involves the topological classification in term of network parameters, but also reveals the consistent picture of intrinsic functional dynamics. Current dynamical analysis focuses on a plethora of functional phenomena: the process of allosteric communication, the binding induced conformational changes, prediction and identification of binding sites of protein complexes, which will give insights into intra-protein complexes interactions. Furthermore, such computational results may elucidate a variety of known biological processes and experimental data, and thereby demonstrate a huge potential for applications such as drug design and functional genomics. Finally we describe some web-based resources for protein complexes, as well as protein network servers and related bioinformatics tools.

  10. The CDC2-related kinase PITALRE is the catalytic subunit of active multimeric protein complexes.

    PubMed Central

    Garriga, J; Mayol, X; Graña, X

    1996-01-01

    PITALRE is a human protein kinase identified by means of its partial sequence identity to the cell division cycle regulatory kinase CDC2. Immunopurified PITALRE protein complexes exhibit an in vitro kinase activity that phosphorylates the retinoblastoma protein, suggesting that PITALRE catalyses this phosphorylation reaction. However, the presence of other kinases in the immunopurified complex could not be ruled out. In the present work, an inactive mutant of the PITALRE kinase has been used to demonstrate that PITALRE is the catalytic subunit responsible for the PITALRE-complex-associated kinase activity, Ectopic overexpression of PITALRE did not increase the total PITALRE kinase activity in the cell, suggesting that PITALRE is regulated by limiting cellular factor(s). Characterization of the PITALRE-containing protein complexes indicated that most of the cellular PITALRE protein exists as a subunit in at least two different active multimeric complexes. Although monomeric PITALRE is also active in vitro, PITALRE present in multimeric complexes exhibits several-fold higher activity than monomeric PITALRE. In addition, overexpression of PITALRE demonstrated the existence of two new associated proteins of approx. 48 and 98 kDa. Altogether these results suggest that, in contrast to the situation with cyclin-dependent kinases, monomeric PITALRE is active, and that association with other proteins modulates its activity and/or its ability to recognize substrates in vivo. PMID:8870681

  11. Complex coacervates of hyaluronic acid and lysozyme: effect on protein structure and physical stability.

    PubMed

    Water, Jorrit J; Schack, Malthe M; Velazquez-Campoy, Adrian; Maltesen, Morten J; van de Weert, Marco; Jorgensen, Lene

    2014-10-01

    Complex coacervates of hyaluronic acid and lysozyme, a model protein, were formed by ionic interaction using bulk mixing and were characterized in terms of binding stoichiometry and protein structure and stability. The complexes were formed at pH 7.2 at low ionic strength (6mM) and the binding stoichiometry was determined using solution depletion and isothermal titration calorimetry. The binding stoichiometry of lysozyme to hyaluronic acid (870 kDa) determined by solution depletion was found to be 225.9 ± 6.6 mol, or 0.1 bound lysozyme molecules per hyaluronic acid monomer. This corresponded well with that obtained by isothermal titration calorimetry of 0.09 bound lysozyme molecules per hyaluronic acid monomer. The complexation did not alter the secondary structure of lysozyme measured by Fourier-transform infrared spectroscopy overlap analysis and had no significant impact on the Tm of lysozyme determined by differential scanning calorimetry. Furthermore, the protein stability of lysozyme was found to be improved upon complexation during a 12-weeks storage study at room temperature, as shown by a significant increase in recovered protein when complexed (94 ± 2% and 102 ± 5% depending on the polymer-protein weight to weight ratio) compared to 89 ± 2% recovery for uncomplexed protein. This study shows the potential of hyaluronic acid to be used in combination with complex coacervation to increase the physical stability of pharmaceutical protein formulations.

  12. Stability and immunogenicity of hypoallergenic peanut protein-polyphenol complexes during in vitro pepsin digestion.

    PubMed

    Plundrich, Nathalie J; White, Brittany L; Dean, Lisa L; Davis, Jack P; Foegeding, E Allen; Lila, Mary Ann

    2015-07-01

    Allergenic peanut proteins are relatively resistant to digestion, and if digested, metabolized peptides tend to remain large and immunoreactive, triggering allergic reactions in sensitive individuals. In this study, the stability of hypoallergenic peanut protein-polyphenol complexes was evaluated during simulated in vitro gastric digestion. When digested with pepsin, the basic subunit of the peanut allergen Ara h 3 was more rapidly hydrolyzed in peanut protein-cranberry or green tea polyphenol complexes compared to uncomplexed peanut flour. Ara h 2 was also hydrolyzed more quickly in the peanut protein-cranberry polyphenol complex than in uncomplexed peanut flour. Peptides from peanut protein-cranberry polyphenol complexes and peanut protein-green tea polyphenol complexes were substantially less immunoreactive (based on their capacity to bind to peanut-specific IgE from patient plasma) compared to peptides from uncomplexed peanut flour. These results suggest that peanut protein-polyphenol complexes may be less immunoreactive passing through the digestive tract in vivo, contributing to their attenuated allergenicity.

  13. Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells.

    PubMed

    Drastichova, Zdenka; Novotny, Jiri

    2012-08-01

    Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors. At least some types of G-protein α subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists. However, the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol. Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of G(q/11)α protein and to determine their localization in isolated fractions and stability in naïve and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G(11)α protein. We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the G(q/11) protein, which were found to be membrane-bound. Both of these complexes dissociated after prolonged treatment with TRH. Still other G(q/11)α protein complexes of lower molecular weight were determined in the cytosol. These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h) hormone treatment. These results support the notion that a portion of G(q/11)α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor. At the same time, these findings indicate that the redistribution of G(q/11)α is brought about by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the G(q/11)α protein.

  14. Structural Analysis of Protein-RNA Complexes in Solution Using NMR Paramagnetic Relaxation Enhancements.

    PubMed

    Hennig, Janosch; Warner, Lisa R; Simon, Bernd; Geerlof, Arie; Mackereth, Cameron D; Sattler, Michael

    2015-01-01

    Biological activity in the cell is predominantly mediated by large multiprotein and protein-nucleic acid complexes that act together to ensure functional fidelity. Nuclear magnetic resonance (NMR) spectroscopy is the only method that can provide information for high-resolution three-dimensional structures and the conformational dynamics of these complexes in solution. Mapping of binding interfaces and molecular interactions along with the characterization of conformational dynamics is possible for very large protein complexes. In contrast, de novo structure determination by NMR becomes very time consuming and difficult for protein complexes larger than 30 kDa as data are noisy and sparse. Fortunately, high-resolution structures are often available for individual domains or subunits of a protein complex and thus sparse data can be used to define their arrangement and dynamics within the assembled complex. In these cases, NMR can therefore be efficiently combined with complementary solution techniques, such as small-angle X-ray or neutron scattering, to provide a comprehensive description of the structure and dynamics of protein complexes in solution. Particularly useful are NMR-derived paramagnetic relaxation enhancements (PREs), which provide long-range distance restraints (ca. 20Å) for structural analysis of large complexes and also report on conformational dynamics in solution. Here, we describe the use of PREs from sample production to structure calculation, focusing on protein-RNA complexes. On the basis of recent examples from our own research, we demonstrate the utility, present protocols, and discuss potential pitfalls when using PREs for studying the structure and dynamic features of protein-RNA complexes.

  15. Sequence co-evolution gives 3D contacts and structures of protein complexes

    PubMed Central

    Hopf, Thomas A; Schärfe, Charlotta P I; Rodrigues, João P G L M; Green, Anna G; Kohlbacher, Oliver; Sander, Chris; Bonvin, Alexandre M J J; Marks, Debora S

    2014-01-01

    Protein–protein interactions are fundamental to many biological processes. Experimental screens have identified tens of thousands of interactions, and structural biology has provided detailed functional insight for select 3D protein complexes. An alternative rich source of information about protein interactions is the evolutionary sequence record. Building on earlier work, we show that analysis of correlated evolutionary sequence changes across proteins identifies residues that are close in space with sufficient accuracy to determine the three-dimensional structure of the protein complexes. We evaluate prediction performance in blinded tests on 76 complexes of known 3D structure, predict protein–protein contacts in 32 complexes of unknown structure, and demonstrate how evolutionary couplings can be used to distinguish between interacting and non-interacting protein pairs in a large complex. With the current growth of sequences, we expect that the method can be generalized to genome-wide elucidation of protein–protein interaction networks and used for interaction predictions at residue resolution. DOI: http://dx.doi.org/10.7554/eLife.03430.001 PMID:25255213

  16. Restricted dynamics of water around a protein-carbohydrate complex: Computer simulation studies

    NASA Astrophysics Data System (ADS)

    Jana, Madhurima; Bandyopadhyay, Sanjoy

    2012-08-01

    Water-mediated protein-carbohydrate interaction is a complex phenomenon responsible for different biological processes in cellular environment. One of the unexplored but important issues in this area is the role played by water during the recognition process and also in controlling the microscopic properties of the complex. In this study, we have carried out atomistic molecular dynamics simulations of a protein-carbohydrate complex formed between the hyaluronan binding domain of the murine Cd44 protein and the octasaccharide hyaluronan in explicit water. Efforts have been made to explore the heterogeneous influence of the complex on the dynamic properties of water present in different regions around it. It is revealed from our analyses that the heterogeneous dynamics of water around the complex are coupled with differential time scales of formation and breaking of hydrogen bonds at the interface. Presence of a highly rigid thin layer of motionally restricted water molecules bridging the protein and the carbohydrate in the common region of the complex has been identified. Such water molecules are expected to play a crucial role in controlling properties of the complex. Importantly, it is demonstrated that the formation of the protein-carbohydrate complex affects the transverse and longitudinal degrees of freedom of the interfacial water molecules in a heterogeneous manner.

  17. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    PubMed Central

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors α and β, and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor β or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein–protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins. PMID:12515863

  18. Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

  19. Quantification of the HIV transcriptional activator complex in live cells by image-based protein-protein interaction analysis.

    PubMed

    Asamitsu, Kaori; Omagari, Katsumi; Okuda, Tomoya; Hibi, Yurina; Okamoto, Takashi

    2016-07-01

    The virus-encoded Tat protein is essential for HIV transcription in infected cells. The interaction of Tat with the cellular transcription elongation factor P-TEFb (positive transcriptional elongation factor b) containing cyclin T1 (CycT1) and cyclin-dependent kinase 9 (CDK9) is critical for its activity. In this study, we use the Fluoppi (fluorescent-based technology detecting protein-protein interaction) system, which enables the quantification of interactions between biomolecules, such as proteins, in live cells. Quantitative measurement of the molecular interactions among Tat, CycT1 and CDK9 has showed that any third molecule enhances the binding between the other two molecules. These findings suggest that each component of the Tat:P-TEFb complex stabilizes the overall complex, thereby supporting the efficient transcriptional elongation during viral RNA synthesis. These interactions may serve as appropriate targets for novel anti-HIV therapy.

  20. Thermal isoelectric precipitation of alpha-lactalbumin from a whey protein concentrate: Influence of protein-calcium complexation.

    PubMed

    Bramaud, C; Aimar, P; Daufin, G

    1995-07-20

    The selective precipitation of alpha-lactalbumin (alpha-LA) at a pH around its isoelectric point (4.2) under heat treatment is the basis for a fractionation process of whey proteins. As precipitation is a phenomenon dependent on the protein hydrophobicity, and as the release of the tightly bound calcium occurring at pH around 4 modifies the alpha-LA hydrophobicity, the specific role of calcium on isoelectric precipitation is investigated. A study of the extent of alpha-LA precipitation in a whey protein concentrate under various operating conditions of pH, temperature, protein concentration, and calcium content is presented. We propose a mechanism for this phenomenon as a combination of a complexation equilibrium and of an irreversible precipitation, to account for the influence of temperature, alpha-LA concentration total ionic content, and calcium concentration, and also to estimate the complexation equilibrium constant. (c) 1995 John Wiley & Sons, Inc.

  1. Chlorophyll-Protein Complexes from the Red-Tide Dinoflagellate, Gonyaulax polyedra Stein 1

    PubMed Central

    Boczar, Barbara A.; Prezelin, Barbara B.

    1987-01-01

    A comparision of high (330 microeinsteins per meter squared per second) and low (80 microeinsteins per meter squared per second) light grown Gonyaulax polyedra indicated a change in the distribution of chlorophyll a, chlorophyll c2, and peridinin among detergent-soluble chlorophyll-protein complexes. Thylakoid fractions were prepared by sonication and centrifugation. Chlorophyll-protein complexes were solubilized from the membranes with sodium dodecyl sulfate and resolved by Deriphat electrophoresis. Low light cells yielded five distinct chlorophyll-protein complexes (I to V), while only four (I′ to IV′) were evident in preparations of high light cells. Both high molecular weight complexes I and I′ were dominated by chlorophyll a absorption and associated with minor amounts of chlorophyll c. Both complexes II and II′ were chlorophyll a-chlorophyll c2-protein complexes devoid of peridinin and unique to dinoflagellates. The chlorophyll a:c2 molar ratio of both complexes was 1:3, indicating significant chlorophyll c enrichment over thylakoid membrane chlorophyll a:c ratios of 1.8 to 2:1. Low light complex III differed from all other high or low light complexes in that it possessed peridinin and had a chlorophyll a:c2 ratio of 1:1. Low light complexes IV and V and high light complexes III′ and IV′ were spectrally similar, had high chlorophyll a:c2 ratios (4:1), and were associated with peridinin. The effects of growth irradiance on the composition of chlorophyll-protein complexes in Gonyaulax polyedra differed from those described for other chlorophyll c-containing plant species. PMID:16665343

  2. Dissecting the molecular organization of the translocon-associated protein complex

    PubMed Central

    Pfeffer, Stefan; Dudek, Johanna; Schaffer, Miroslava; Ng, Bobby G.; Albert, Sahradha; Plitzko, Jürgen M.; Baumeister, Wolfgang; Zimmermann, Richard; Freeze, Hudson H.; Engel, Benjamin D.; Förster, Friedrich

    2017-01-01

    In eukaryotic cells, one-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon. The translocon-associated protein (TRAP) complex is an integral component of the translocon, assisting the Sec61 protein-conducting channel by regulating signal sequence and transmembrane helix insertion in a substrate-dependent manner. Here we use cryo-electron tomography (CET) to study the structure of the native translocon in evolutionarily divergent organisms and disease-linked TRAP mutant fibroblasts from human patients. The structural differences detected by subtomogram analysis form a basis for dissecting the molecular organization of the TRAP complex. We assign positions to the four TRAP subunits within the complex, providing insights into their individual functions. The revealed molecular architecture of a central translocon component advances our understanding of membrane protein biogenesis and sheds light on the role of TRAP in human congenital disorders of glycosylation. PMID:28218252

  3. On the importance of polar interactions for complexes containing intrinsically disordered proteins.

    PubMed

    Wong, Eric T C; Na, Dokyun; Gsponer, Jörg

    2013-01-01

    There is a growing recognition for the importance of proteins with large intrinsically disordered (ID) segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions.

  4. Analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes.

    PubMed

    Tetlow, Ian J; Beisel, Kim G; Cameron, Scott; Makhmoudova, Amina; Liu, Fushan; Bresolin, Nicole S; Wait, Robin; Morell, Matthew K; Emes, Michael J

    2008-04-01

    Protein-protein interactions among enzymes of amylopectin biosynthesis were investigated in developing wheat (Triticum aestivum) endosperm. Physical interactions between starch branching enzymes (SBEs) and starch synthases (SSs) were identified from endosperm amyloplasts during the active phase of starch deposition in the developing grain using immunoprecipitation and cross-linking strategies. Coimmunoprecipitation experiments using peptide-specific antibodies indicate that at least two distinct complexes exist containing SSI, SSIIa, and either of SBEIIa or SBEIIb. Chemical cross linking was used to identify protein complexes containing SBEs and SSs from amyloplast extracts. Separation of extracts by gel filtration chromatography demonstrated the presence of SBE and SS forms in protein complexes of around 260 kD and that SBEII forms may also exist as homodimers. Analysis of cross-linked 260-kD aggregation products from amyloplast lysates by mass spectrometry confirmed SSI, SSIIa, and SBEII forms as components of one or more protein complexes in amyloplasts. In vitro phosphorylation experiments with gamma-(32)P-ATP indicated that SSII and both forms of SBEII are phosphorylated. Treatment of the partially purified 260-kD SS-SBE complexes with alkaline phosphatase caused dissociation of the assembly into the respective monomeric proteins, indicating that formation of SS-SBE complexes is phosphorylation dependent. The 260-kD SS-SBEII protein complexes are formed around 10 to 15 d after pollination and were shown to be catalytically active with respect to both SS and SBE activities. Prior to this developmental stage, SSI, SSII, and SBEII forms were detectable only in monomeric form. High molecular weight forms of SBEII demonstrated a higher affinity for in vitro glucan substrates than monomers. These results provide direct evidence for the existence of protein complexes involved in amylopectin biosynthesis.

  5. Measurement of protein HC (alpha 1 microglobulin) and protein HC-IgA complex in different body fluids.

    PubMed Central

    Fernández-Luna, J L; Leyva-Cobián, F; Méndez, E

    1988-01-01

    Protein HC and protein HC-IgA complex were measured in 18 different types of fluid sample from healthy subjects and patients with different illnesses to determine if the concentrations of protein HC and protein HC-IgA complexes could be used to monitor certain diseases, when measured separately. The normal values for HC ranged from between 0.30 mg/l in saliva and 11.7 mg/l in blood plasma. HC-IgA complex has a greater range, from undetectable concentrations (urine, colostrum, and cervical mucus) up to 59.2 mg/l in blood plasma. Undetectable concentrations of HC-IgA complex were also shown in serum from patients with IgA immune deficiency and in cerebrospinal fluid from patients with multiple sclerosis. Increased concentrations of HC were noted in bronchoalveolar fluid from a patient with pulmonary alveolar proteinosis, serum from patients with Behcet's syndrome, and in synovial fluid from patients with gout, chondrocalcinosis, and rheumatoid arthritis. On the other hand, the concentrations of HC-IgA complex were raised only in those patients with pulmonary alveolar proteinosis or rheumatoid arthritis. PMID:2463270

  6. Contribution of Human Oral Cells to Astringency by Binding Salivary Protein/Tannin Complexes.

    PubMed

    Soares, Susana; Ferrer-Galego, Raúl; Brandão, Elsa; Silva, Mafalda; Mateus, Nuno; Freitas, Victor de

    2016-10-10

    The most widely accepted mechanism to explain astringency is the interaction and precipitation of salivary proteins by food tannins, in particular proline-rich proteins. However, other mechanisms have been arising to explain astringency, such as binding of tannins to oral cells. In this work, an experimental method was adapted to study the possible contribution of both salivary proteins and oral cells to astringency induced by grape seed procyanidin fractions. Overall, in the absence of salivary proteins, the extent of procyanidin complexation with oral cells increased with increasing procyanidin degree of polymerization (mDP). Procyanidin fractions rich in monomers were the ones with the lowest ability to bind to oral cells. In the presence of salivary proteins and for procyanidins with mDP 2 the highest concentrations (1.5 and 2.0 mM) resulted in an increased binding of procyanidins to oral cells. This was even more evident for fractions III and IV at 1.0 mM and upper concentrations. Regarding the salivary proteins affected, it was possible to observe a decrease of P-B peptide and aPRP proteins for fractions II and III. This decrease is greater as the procyanidins' mDP increases. In fact, for fraction IV an almost total depletion of all salivary proteins was observed. This decrease is due to the formation of insoluble salivary protein/procyanidin complexes. Altogether, these data suggest that some procyanidins are able to bind to oral cells and that the salivary proteins interact with procyanidins forming salivary protein/procyanidin complexes that are also able to link to oral cells. The procyanidins that remain unbound to oral cells are able to bind to salivary proteins forming a large network of salivary protein/procyanidin complexes. Overall, the results presented herein provide one more step to understand food oral astringency onset.

  7. Identification of low-abundance proteins in serum via the isolation of HSP72 complexes.

    PubMed

    Tanaka, Masako; Shiota, Masayuki; Nakao, Takafumi; Uemura, Ryo; Nishi, Satoshi; Ohkawa, Yasuyuki; Matsumoto, Masaki; Yamaguchi, Maki; Osada-Oka, Mayuko; Inagaki, Azusa; Takahashi, Katsuyuki; Nakayama, Keiichi I; Gi, Min; Izumi, Yasukatsu; Miura, Katsuyuki; Iwao, Hiroshi

    2016-03-16

    Heat shock protein 72 (HSP72) is an intracellular molecular chaperone that is overexpressed in tumor cells, and has also been detected in extracellular regions such as the blood. HSP72 forms complexes with peptides and proteins that are released from tumors. Accordingly, certain HSP72-binding proteins/peptides present in the blood of cancer patients may be derived from tumor cells. In this study, to effectively identify low-abundance proteins/peptides in the blood as tumor markers, we established a method for isolating HSP72-binding proteins/peptides from serum. Nine HSP72-specific monoclonal antibodies were conjugated to N-hydroxysulfosuccinimide-activated Sepharose beads (NHq) and used to isolate HSP72 complexes from serum samples. Precipitated proteins were then identified by LC-MS/MS analysis. Notably, this approach enabled the isolation of low-abundance proteins from serum without albumin removal. Moreover, by subjecting the serum samples of ten patients with multiple myeloma (MM) to NHq analysis, we identified 299 proteins present in MM HSP72 complexes, including 65 intracellular proteins. Among the intracellular proteins detected, 21 were present in all serum samples tested, while 11 were detected in both the conditioned media from cultured multiple myeloma cells and serum from MM patients. These results suggest that the NHq method can be applied to discover candidate tumor markers.

  8. Conservation and Variability of Synaptonemal Complex Proteins in Phylogenesis of Eukaryotes

    PubMed Central

    Grishaeva, Tatiana M.; Bogdanov, Yuri F.

    2014-01-01

    The problems of the origin and evolution of meiosis include the enigmatic variability of the synaptonemal complexes (SCs) which, being morphology similar, consist of different proteins in different eukaryotic phyla. Using bioinformatics methods, we monitored all available eukaryotic proteomes to find proteins similar to known SC proteins of model organisms. We found proteins similar to SC lateral element (LE) proteins and possessing the HORMA domain in the majority of the eukaryotic taxa and assume them the most ancient among all SC proteins. Vertebrate LE proteins SYCP2, SYCP3, and SC65 proved to have related proteins in many invertebrate taxa. Proteins of SC central space are most evolutionarily variable. It means that different protein-protein interactions can exist to connect LEs. Proteins similar to the known SC proteins were not found in Euglenophyta, Chrysophyta, Charophyta, Xanthophyta, Dinoflagellata, and primitive Coelomata. We conclude that different proteins whose common feature is the presence of domains with a certain conformation are involved in the formation of the SC in different eukaryotic phyla. This permits a targeted search for orthologs of the SC proteins using phylogenetic trees. Here we consider example of phylogenetic trees for protozoans, fungi, algae, mosses, and flowering plants. PMID:25147749

  9. Conservation and variability of synaptonemal complex proteins in phylogenesis of eukaryotes.

    PubMed

    Grishaeva, Tatiana M; Bogdanov, Yuri F

    2014-01-01

    The problems of the origin and evolution of meiosis include the enigmatic variability of the synaptonemal complexes (SCs) which, being morphology similar, consist of different proteins in different eukaryotic phyla. Using bioinformatics methods, we monitored all available eukaryotic proteomes to find proteins similar to known SC proteins of model organisms. We found proteins similar to SC lateral element (LE) proteins and possessing the HORMA domain in the majority of the eukaryotic taxa and assume them the most ancient among all SC proteins. Vertebrate LE proteins SYCP2, SYCP3, and SC65 proved to have related proteins in many invertebrate taxa. Proteins of SC central space are most evolutionarily variable. It means that different protein-protein interactions can exist to connect LEs. Proteins similar to the known SC proteins were not found in Euglenophyta, Chrysophyta, Charophyta, Xanthophyta, Dinoflagellata, and primitive Coelomata. We conclude that different proteins whose common feature is the presence of domains with a certain conformation are involved in the formation of the SC in different eukaryotic phyla. This permits a targeted search for orthologs of the SC proteins using phylogenetic trees. Here we consider example of phylogenetic trees for protozoans, fungi, algae, mosses, and flowering plants.

  10. Role of Ca²⁺ in folding the tandem β-sandwich extender domains of a bacterial ice-binding adhesin.

    PubMed

    Guo, Shuaiqi; Garnham, Christopher P; Karunan Partha, Sarathy; Campbell, Robert L; Allingham, John S; Davies, Peter L

    2013-11-01

    A Ca(2+) -dependent 1.5-MDa antifreeze protein present in an Antarctic Gram-negative bacterium, Marinomonas primoryensis (MpAFP), has recently been reassessed as an ice-binding adhesin. The non-ice-binding region II (RII), one of five distinct domains in MpAFP, constitutes ~ 90% of the protein. RII consists of ~ 120 tandem copies of an identical 104-residue sequence. We used the Protein Homology/analogy Recognition Engine server to define the boundaries of a single 104-residue RII construct (RII monomer). CD demonstrated that Ca(2+) is required for RII monomer folding, and that the monomer is fully structured at a Ca(2+) /protein molar ratio of 10 : 1. The crystal structure of the RII monomer was solved to a resolution of 1.35 Å by single-wavelength anomalous dispersion and molecular replacement methods with Ca(2+) as the heavy atom to obtain phase information. The RII monomer folds as a Ca(2+) -bound immunoglobulin-like β-sandwich. Ca(2+) ions are coordinated at the interfaces between each RII monomer and its symmetry-related molecules, suggesting that these ions may be involved in the stabilization of the tandemly repeated RII. We hypothesize that > 600 Ca(2+) ions help to rigidify the chain of 104-residue repeats in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed role of RII is to help the strictly aerobic bacterium bind surface ice in an Antarctic lake for better access to oxygen and nutrients. This work may give insights into other bacterial proteins that resemble MpAFP, especially those of the large repeats-in-toxin family that have been characterized as adhesins exported via the type I secretion pathway.

  11. Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125.

    PubMed

    Walden, Miriam; Crow, Allister; Nelson, Miles D; Banfield, Mark J

    2014-03-01

    Streptococcus pyogenes and other Gram-positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild-type protein and a set of mutants, we assessed the proteolytic and thermal stability of the S. pyogenes pilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125(Cys426Ala). We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands.

  12. The EPA2 adhesin encoding gene is responsive to oxidative stress in the opportunistic fungal pathogen Candida glabrata.

    PubMed

    Juárez-Cepeda, Jacqueline; Orta-Zavalza, Emmanuel; Cañas-Villamar, Israel; Arreola-Gómez, Jorge; Pérez-Cornejo, Gloria Patricia; Hernández-Carballo, Carmen Yudith; Gutiérrez-Escobedo, Guadalupe; Castaño, Irene; De Las Peñas, Alejandro

    2015-11-01

    Candida glabrata has emerged as an important opportunistic pathogen in both mucosal and bloodstream infections. C. glabrata contains 67 adhesin-like glycosylphosphatidylinositol-cell-wall proteins (GPI-CWPs), which are classified into seven groups and the largest is the Epa family. Epa proteins are very diverse and their expression is differentially regulated. Like many of the EPA genes, EPA2 is localized in a subtelomeric region where it is subject to chromatin-based transcriptional silencing and its role remains largely unexplored. In this study, we show that EPA2 gene is induced specifically in vitro in the presence of oxidative stress generated by H2O2. This induction is dependent on both Yap1 and Skn7, whereas Msn4 represses EPA2 expression. Interestingly, EPA2 is not induced during phagocytosis, but its expression can be identified in the liver in a murine model of systemic infection. Epa2 has no effect on the virulence of C. glabrata. The work presented herein provides a foundation for future studies to dissect the molecular mechanism(s) by which EPA2 of C. glabrata can be induced in the presence of oxidative stress in a region subject to subtelomeric silencing.

  13. Visualization of recombinant DNA and protein complexes using atomic force microscopy.

    PubMed

    Murphy, Patrick J M; Shannon, Morgan; Goertz, John

    2011-07-18

    Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1). Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation, such as Ni(2+) or Mg(2+), which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase and a repair enzyme, bound to individual DNA strands. These experiments provide

  14. Application of model bread baking in the examination of arabinoxylan-protein complexes in rye bread.

    PubMed

    Buksa, Krzysztof

    2016-09-05

    The changes in molecular mass of arabinoxylan (AX) and protein caused by bread baking process were examined using a model rye bread. Instead of the normal flour, the dough contained starch, water-extractable AX and protein which were isolated from rye wholemeal. From the crumb of selected model breads, starch was removed releasing AX-protein complexes, which were further examined by size exclusion chromatography. On the basis of the research, it was concluded that optimum model mix can be composed of 3-6% AX and 3-6% rye protein isolate at 94-88% of rye starch meaning with the most similar properties to low extraction rye flour. Application of model rye bread allowed to examine the interactions between AX and proteins. Bread baked with a share of AX, rye protein and starch, from which the complexes of the highest molar mass were isolated, was characterized by the strongest structure of the bread crumb.

  15. Probing protein complexes inside living cells using a silicon nanowire-based pull-down assay

    NASA Astrophysics Data System (ADS)

    Choi, Sojoong; Kim, Hyunju; Kim, So Yeon; Yang, Eun Gyeong

    2016-06-01

    Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells.Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells. Electronic supplementary information (ESI) available: Materials, experimental methods and Fig. S1-S8. See DOI: 10.1039/c6nr00171h

  16. An Ocular Protein Triad Can Classify Four Complex Retinal Diseases

    PubMed Central

    Kuiper, J. J. W.; Beretta, L.; Nierkens, S.; van Leeuwen, R.; ten Dam-van Loon, N. H.; Ossewaarde-van Norel, J.; Bartels, M. C.; de Groot-Mijnes, J. D. F.; Schellekens, P.; de Boer, J. H.; Radstake, T. R. D. J.

    2017-01-01

    Retinal diseases generally are vision-threatening conditions that warrant appropriate clinical decision-making which currently solely dependents upon extensive clinical screening by specialized ophthalmologists. In the era where molecular assessment has improved dramatically, we aimed at the identification of biomarkers in 175 ocular fluids to classify four archetypical ocular conditions affecting the retina (age-related macular degeneration, idiopathic non-infectious uveitis, primary vitreoretinal lymphoma, and rhegmatogenous retinal detachment) with one single test. Unsupervised clustering of ocular proteins revealed a classification strikingly similar to the clinical phenotypes of each disease group studied. We developed and independently validated a parsimonious model based merely on three proteins; interleukin (IL)-10, IL-21, and angiotensin converting enzyme (ACE) that could correctly classify patients with an overall accuracy, sensitivity and specificity of respectively, 86.7%, 79.4% and 92.5%. Here, we provide proof-of-concept for molecular profiling as a diagnostic aid for ophthalmologists in the care for patients with retinal conditions. PMID:28128370

  17. An Ocular Protein Triad Can Classify Four Complex Retinal Diseases

    NASA Astrophysics Data System (ADS)

    Kuiper, J. J. W.; Beretta, L.; Nierkens, S.; van Leeuwen, R.; Ten Dam-van Loon, N. H.; Ossewaarde-van Norel, J.; Bartels, M. C.; de Groot-Mijnes, J. D. F.; Schellekens, P.; de Boer, J. H.; Radstake, T. R. D. J.

    2017-01-01

    Retinal diseases generally are vision-threatening conditions that warrant appropriate clinical decision-making which currently solely dependents upon extensive clinical screening by specialized ophthalmologists. In the era where molecular assessment has improved dramatically, we aimed at the identification of biomarkers in 175 ocular fluids to classify four archetypical ocular conditions affecting the retina (age-related macular degeneration, idiopathic non-infectious uveitis, primary vitreoretinal lymphoma, and rhegmatogenous retinal detachment) with one single test. Unsupervised clustering of ocular proteins revealed a classification strikingly similar to the clinical phenotypes of each disease group studied. We developed and independently validated a parsimonious model based merely on three proteins; interleukin (IL)-10, IL-21, and angiotensin converting enzyme (ACE) that could correctly classify patients with an overall accuracy, sensitivity and specificity of respectively, 86.7%, 79.4% and 92.5%. Here, we provide proof-of-concept for molecular profiling as a diagnostic aid for ophthalmologists in the care for patients with retinal conditions.

  18. Protein complex analysis of native brain potassium channels by proteomics.

    PubMed

    Sandoz, Guillaume; Lesage, Florian

    2008-01-01

    TREK potassium channels belong to a family of channel subunits with two-pore domains (K(2P)). TREK1 knockout mice display impaired polyunsaturated fatty acid-mediated protection against brain ischemia, reduced sensitivity to volatile anesthetics, resistance to depression and altered perception of pain. Recently, we isolated native TREK1 channels from mouse brain and identified their specific components by mass spectrometry. Among the identified partners, the A-Kinase Anchoring Protein AKAP150 binds to a regulatory domain of TREK1 and acts as a molecular switch. It transforms low activity, outwardly rectifying TREK1 currents into robust leak conductances resistant to stimulation by arachidonic acid, membrane stretch and acidification. Inhibition of the TREK1/AKAP150 channel by Gs-coupled receptors is as extensive as for TREK1 alone (but faster) whereas inhibition of TREK1/AKAP150 by Gq-coupled receptors is reduced. Furthermore, the association of AKAP150 with TREK1 channels integrates them into postsynaptic scaffolds where G protein-coupled membrane receptors and channels dock simultaneously. This chapter describes the proteomic approach used to study the composition of native TREK1 channels and point out its advantages and limitations over more classical methods (two-hybrid screenings in the yeast and bacteria or GST-pull down).

  19. Protein-free parallel triple-stranded DNA complex formation

    PubMed Central

    Shchyolkina, A. K.; Timofeev, E. N.; Lysov, Yu. P.; Florentiev, V. L.; Jovin, T. M.; Arndt-Jovin, D. J.

    2001-01-01

    A 14 nt DNA sequence 5′-AGAATGTGGCAAAG-3′ from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar–phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5′-end of the probe strand as donor and BODIPY-Texas Red on the 3′-amino group of either strand of the target duplex as acceptor. There was full protection from OsO4-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe–intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed. PMID:11160932

  20. Novel UV assay for protein determination and the characterization of copper-protein complexes by mass spectrometry.

    PubMed

    Drochioiu, G; Damoc, N E; Przybylski, M

    2006-05-15

    A very simple, highly selective and sensitive assay of proteins based on the biuret absorption in the ultraviolet region has been developed. The well-known biuret assay is based on the reaction of proteins with copper ions under strong alkaline conditions to form a copper-protein complex. Yet, copper ions may seriously interfere with the determination if the measurement is made in the UV range. In the present approach, proteins mobilize copper ions from insoluble salts at different pH values, and the copper-protein complexes are investigated by UV spectrophotometry and mass spectrometry. Upon using copper phosphate, free copper ions do not interfere with the determination from 540 to 240nm. Copper absorbance slowly increases from 240 to 190nm where a blank with the reagents is recommended. A maximum absorbance for the copper-protein complex was found at 226nm and high pH value. The stoichiometries of the copper-protein complexes measured directly with a mass spectrometer are pH dependent: half of the peptides without any histidine residue chelate just a single Cu(2+) ion at pH 7.4 but each such peptide mobilizes from 1 to 6 Cu(2+) ions at pH 10.3. To determine proteins, 1-1.5ml of 1.8% KOH solution with 0-20mugml(-1) protein is treated with 25mg of copper phosphate powder. The mixture is powerfully stirred, centrifuged, and the absorbance of the supernatant is measured at 226nm in 1cm quartz cuvettes against a blank of the reagents. The color system obeys Beer's law in the range 0.1-20mugml(-1) protein at this wavelength. The molar absorptivity value proved to be a characteristic of each protein being analyzed. Therefore, individual proteins should be used to plot calibration curves. This assay proved to be over 100 times more sensitive than the classical biuret procedure. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of

  1. Antibodies derived from an enterotoxigenic Escherichia coli (ETEC) adhesin tip MEFA (multiepitope fusion antigen) against adherence of nine ETEC adhesins: CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA.

    PubMed

    Nandre, Rahul M; Ruan, Xiaosai; Duan, Qiangde; Sack, David A; Zhang, Weiping

    2016-06-30

    Diarrhea continues to be a leading cause of death in children younger than 5 years in developing countries. Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of children's diarrhea and travelers' diarrhea. ETEC bacteria initiate diarrheal disease by attaching to host receptors at epithelial cells and colonizing in small intestine. Therefore, preventing ETEC attachment has been considered the first line of defense against ETEC diarrhea. However, developing vaccines effectively against ETEC bacterial attachment encounters challenge because ETEC strains produce over 23 immunologically heterogeneous adhesins. In this study, we applied MEFA (multiepitope fusion antigen) approach to integrate epitopes from adhesin tips or adhesive subunits of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA adhesins and to construct an adhesin tip MEFA peptide. We then examined immunogenicity of this tip MEFA in mouse immunization, and assessed potential application of this tip MEFA for ETEC vaccine development. Data showed that mice intraperitoneally immunized with this adhesin tip MEFA developed IgG antibody responses to all nine ETEC adhesins. Moreover, ETEC and E. coli bacteria expressing these nine adhesins, after incubation with serum of the immunized mice, exhibited significant reduction in attachment to Caco-2 cells. These results indicated that anti-adhesin antibodies induced by this adhesin tip MEFA blocked adherence of the most important ETEC adhesins, suggesting this multivalent tip MEFA may be useful for developing a broadly protective anti-adhesin vaccine against ETEC diarrhea.

  2. Vaccination with a recombinant fragment of collagen adhesin provides protection against Staphylococcus aureus-mediated septic death.

    PubMed Central

    Nilsson, I M; Patti, J M; Bremell, T; Höök, M; Tarkowski, A

    1998-01-01

    Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. Morbidity and mortality due to infections such as sepsis, osteomyelitis, septic arthritis, and invasive endocarditis remain high despite the use of antibiotics. The emergence of antibiotic resistant super bugs mandates that alternative strategies for the prevention and treatment of S. aureus infections are developed. We investigated the ability of vaccination with a recombinant fragment of the S. aureus collagen adhesin to protect mice against sepsis-induced death. Actively immunized NMRI mice were intravenously inoculated with the S. aureus clinical isolate strain Phillips. 14 d after inoculation, mortality in the collagen adhesin-vaccinated group was only 13%, compared with 87% in the control antigen immunized group (P < 0.001). To determine if the protective effect was antibody mediated, we passively immunized naive mice with collagen adhesin-specific antibodies. Similar to the active immunization strategy, passive transfer of collagen adhesin-specific antibodies protected mice against sepsis-induced death. In vitro experiments indicated that S. aureus opsonized with sera from collagen adhesin immunized mice promoted phagocytic uptake and enhanced intracellular killing compared with bacteria opsonized with sera from control animals. These results indicate that the collagen adhesin is a viable target in the development of immunotherapeutics against S. aureus. PMID:9637697

  3. An informatic framework for decoding protein complexes by top-down mass spectrometry

    PubMed Central

    Skinner, Owen S.; Havugimana, Pierre C.; Haverland, Nicole A.; Fornelli, Luca; Early, Bryan P.; Greer, Joseph B.; Fellers, Ryan T.; Durbin, Kenneth R.; Do Vale, Luis H. F.; Melani, Rafael D.; Seckler, Henrique S.; Nelp, Micah T.; Belov, Mikhail E.; Horning, Stevan R.; Makarov, Alexander A.; LeDuc, Richard D.; Bandarian, Vahe; Compton, Philip D.; Kelleher, Neil L.

    2015-01-01

    Efforts to map the human protein interactome have resulted in information about hundreds to thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we combined the CORUM and UniProt databases to create candidates for an error-tolerant search engine designed for hierarchical top-down analyses, identification, and scoring of multi-proteoform complexes by native mass spectrometry. PMID:26780093

  4. Folding and association of a homotetrameric protein complex in an all-atom Go model.

    PubMed

    Berhanu, W M; Jiang, P; Hansmann, U H E

    2013-01-01

    The 84-residue homotetrameric BBAT1 is one of the smallest stable protein complexes and therefore is a good test system to study the self-assembly of multimeric proteins. We have researched for this protein the interplay between the folding of monomers and their assembly into tetramers. Replica exchange molecular dynamics simulations relying on a Go model are compared with earlier simulations that use the physics-based coarse-grained UNRES model.

  5. The biological activities of protein/oleic acid complexes reside in the fatty acid.

    PubMed

    Fontana, Angelo; Spolaore, Barbara; Polverino de Laureto, Patrizia

    2013-06-01

    A complex formed by human α-lactalbumin (α-LA) and oleic acid (OA), named HAMLET, has been shown to have an apoptotic activity leading to the selective death of tumor cells. In numerous publications it has been reported that in the complex α-LA is monomeric and adopts a partly folded or "molten globule" state, leading to the idea that partly folded proteins can have "beneficial effects". The protein/OA molar ratio initially has been reported to be 1:1, while recent data have indicated that the OA-complex is given by an oligomeric protein capable of binding numerous OA molecules per protein monomer. Proteolytic fragments of α-LA, as well as other proteins unrelated to α-LA, can form OA-complexes with biological activities similar to those of HAMLET, thus indicating that a generic protein can form a cytotoxic complex under suitable experimental conditions. Moreover, even the selective tumoricidal activity of HAMLET-like complexes has been questioned. There is recent evidence that the biological activity of long chain unsaturated fatty acids, including OA, can be ascribed to their effect of perturbing the structure of biological membranes and consequently the function of membrane-bound proteins. In general, it has been observed that the cytotoxic effects exerted by HAMLET-like complexes are similar to those reported for OA alone. Overall, these findings can be interpreted by considering that the protein moiety does not have a toxic effect on its own, but merely acts as a solubilising agent for the inherently toxic fatty acid.

  6. A machine learning approach for ranking clusters of docked protein-protein complexes by pairwise cluster comparison.

    PubMed

    Pfeiffenberger, Erik; Chaleil, Raphael A G; Moal, Iain H; Bates, Paul A

    2017-03-01

    Reliable identification of near-native poses of docked protein-protein complexes is still an unsolved problem. The intrinsic heterogeneity of protein-protein interactions is challenging for traditional biophysical or knowledge based potentials and the identification of many false positive binding sites is not unusual. Often, ranking protocols are based on initial clustering of docked poses followed by the application of an energy function to rank each cluster according to its lowest energy member. Here, we present an approach of cluster ranking based not only on one molecular descriptor (e.g., an energy function) but also employing a large number of descriptors that are integrated in a machine learning model, whereby, an extremely randomized tree classifier based on 109 molecular descriptors is trained. The protocol is based on first locally enriching clusters with additional poses, the clusters are then characterized using features describing the distribution of molecular descriptors within the cluster, which are combined into a pairwise cluster comparison model to discriminate near-native from incorrect clusters. The results show that our approach is able to identify clusters containing near-native protein-protein complexes. In addition, we present an analysis of the descriptors with respect to their power to discriminate near native from incorrect clusters and how data transformations and recursive feature elimination can improve the ranking performance. Proteins 2017; 85:528-543. © 2016 Wiley Periodicals, Inc.

  7. Identification of Chlamydia trachomatis outer membrane complex proteins by differential proteomics.

    PubMed

    Liu, Xiaoyun; Afrane, Mary; Clemmer, David E; Zhong, Guangming; Nelson, David E

    2010-06-01

    The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra- and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses of Chlamydia trachomatis serovar L2 434/Bu EB, COMC, and Sarkosyl-soluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.

  8. Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles

    PubMed Central

    Newell-Litwa, Karen; Salazar, Gloria; Smith, Yoland

    2009-01-01

    Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both selected synaptic vesicle and lysosomal membrane proteins coexist with the adaptor protein complex 3 (AP-3) in neuronal cells. From these early endosomes, both synaptic vesicle membrane proteins and characteristic AP-3 lysosomal cargoes can be similarly sorted to brain synaptic vesicles and PC12 synaptic-like microvesicles. Mouse knockouts for two Hermansky–Pudlak complexes involved in lysosomal biogenesis from early endosomes, the ubiquitous isoform of AP-3 (Ap3b1−/−) and muted, defective in the biogenesis of lysosome-related organelles complex 1 (BLOC-1), increased the content of characteristic synaptic vesicle proteins and known AP-3 lysosomal proteins in isolated synaptic vesicle fractions. These phenotypes contrast with those of the mouse knockout for the neuronal AP-3 isoform involved in synaptic vesicle biogenesis (Ap3b2−/−), in which the content of select proteins was reduced in synaptic vesicles. Our results demonstrate that lysosomal and lysosome-related organelle biogenesis mechanisms regulate steady-state synaptic vesicle protein composition from shared early endosomes. PMID:19144828

  9. Protein adsorption induced bridging flocculation: the dominant entropic pathway for nano-bio complexation

    NASA Astrophysics Data System (ADS)

    Eren, Necla Mine; Narsimhan, Ganesan; Campanella, Osvaldo H.

    2016-02-01

    Lysozyme-silica interactions and the resulting complexation were investigated through adsorption isotherms, dynamic and electrophoretic light scattering, circular dichroism (CD), and isothermal titration calorimetry (ITC). A thermodynamic analysis of ITC data revealed the existence of two binding modes during protein-nanoparticle complexation. Both binding modes are driven by the cooperation of a favorable enthalpy in the presence of a dominating entropy gain. The first binding mode has a higher binding affinity, a lower equilibrium stoichiometry and is driven by a higher entropic contribution compared to the second type. The observed favorable enthalpy gain in both modes is attributed to non-covalent complexation whereas the entropy gain is associated with the re-organization of the silica surface including not only the solvent and counter ion release, but also the protein's conformational changes. Possible mechanisms are proposed to explain non-covalent complexations for each binding mode by relating the changes in the zeta potential and hydrodynamic radius to the obtained adsorption isotherms and calorimetry profile. Based on all these findings, it is proposed that lysozyme adsorption on nano-silica is the result of protein-nanoparticle and protein-protein interactions that further leads to spontaneous, non-directional and random complexation of silica through bridging flocculation.Lysozyme-silica interactions and the resulting complexation were investigated through adsorption isotherms, dynamic and electrophoretic light scattering, circular dichroism (CD), and isothermal titration calorimetry (ITC). A thermodynamic analysis of ITC data revealed the existence of two binding modes during protein-nanoparticle complexation. Both binding modes are driven by the cooperation of a favorable enthalpy in the presence of a dominating entropy gain. The first binding mode has a higher binding affinity, a lower equilibrium stoichiometry and is driven by a higher entropic

  10. Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

    PubMed

    Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

    2014-12-11

    The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

  11. Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

    PubMed Central

    Sommer, Maik S.; Schleiff, Enrico

    2014-01-01

    With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity. PMID:25085907

  12. Programming Controlled Adhesion of E. coli to Target Surfaces, Cells, and Tumors with Synthetic Adhesins

    PubMed Central

    2014-01-01

    In this work we report synthetic adhesins (SAs) enabling the rational design of the adhesion properties of E. coli. SAs have a modular structure comprising a stable β-domain for outer membrane anchoring and surface-exposed immunoglobulin domains with high affinity and specificity that can be selected from large repertoires. SAs are constitutively and stably expressed in an E. coli strain lacking a conserved set of natural adhesins, directing a robust, fast, and specific adhesion of bacteria to target antigenic surfaces and cells. We demonstrate the functionality of SAs in vivo, showing that, compared to wild type E. coli, lower doses of engineered E. coli are sufficient to colonize solid tumors expressing an antigen recognized by the SA. In addition, lower levels of engineered bacteria were found in non-target tissues. Therefore, SAs provide stable and specific adhesion capabilities to E. coli against target surfaces of interest for diverse applications using live bacteria. PMID:25045780

  13. Bacteriophage adhesin-coated long-period gratings for bacterial lipopolysaccharide recognition

    NASA Astrophysics Data System (ADS)

    Koba, Marcin; Śmietana, Mateusz; Brzozowska, Ewa; Górska, Sabina; Mikulic, Predrag; Bock, Wojtek J.

    2014-05-01

    In this work we report an application of the optical fiber long-period gratings (LPGs) working near the dispersion turning point of higher order cladding modes for bacterial lipopolysaccharide (LPS) recognition. We show that when the LPG is functionalized with the bacteriophage adhesin, it is capable of very specific LPS detection. Thus, we compare label-free binding effect for specific to the adhesin LPS-positive and non-specific LPS-negative. The resonance wavelength shift induced by the LPS-positive reaches 2.9 nm, while for LPS-negative the shift is negligible. The LPG-based sensing structure allows for monitoring of the binding phenomenon in real time and with good accuracy.

  14. Programming controlled adhesion of E. coli to target surfaces, cells, and tumors with synthetic adhesins.

    PubMed

    Piñero-Lambea, Carlos; Bodelón, Gustavo; Fernández-Periáñez, Rodrigo; Cuesta, Angel M; Álvarez-Vallina, Luis; Fernández, Luis Ángel

    2015-04-17

    In this work we report synthetic adhesins (SAs) enabling the rational design of the adhesion properties of E. coli. SAs have a modular structure comprising a stable β-domain for outer membrane anchoring and surface-exposed immunoglobulin domains with high affinity and specificity that can be selected from large repertoires. SAs are constitutively and stably expressed in an E. coli strain lacking a conserved set of natural adhesins, directing a robust, fast, and specific adhesion of bacteria to target antigenic surfaces and cells. We demonstrate the functionality of SAs in vivo, showing that, compared to wild type E. coli, lower doses of engineered E. coli are sufficient to colonize solid tumors expressing an antigen recognized by the SA. In addition, lower levels of engineered bacteria were found in non-target tissues. Therefore, SAs provide stable and specific adhesion capabilities to E. coli against target surfaces of interest for diverse applications using live bacteria.

  15. Protein complex-based analysis framework for high-throughput data sets.

    PubMed

    Vinayagam, Arunachalam; Hu, Yanhui; Kulkarni, Meghana; Roesel, Charles; Sopko, Richelle; Mohr, Stephanie E; Perrimon, Norbert

    2013-02-26

    Analysis of high-throughput data increasingly relies on pathway annotation and functional information derived from Gene Ontology. This approach has limitations, in particular for the analysis of network dynamics over time or under different experimental conditions, in which modules within a network rather than complete pathways might respond and change. We report an analysis framework based on protein complexes, which are at the core of network reorganization. We generated a protein complex resource for human, Drosophila, and yeast from the literature and databases of protein-protein interaction networks, with each species having thousands of complexes. We developed COMPLEAT (http://www.flyrnai.org/compleat), a tool for data mining and visualization for complex-based analysis of high-throughput data sets, as well as analysis and integration of heterogeneous proteomics and gene expression data sets. With COMPLEAT, we identified dynamically regulated protein complexes among genome-wide RNA interference data sets that used the abundance of phosphorylated extracellular signal-regulated kinase in cells stimulated with either insulin or epidermal growth factor as the output. The analysis predicted that the Brahma complex participated in the insulin response.

  16. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

    SciTech Connect

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

  17. p300/CREB Binding Protein-Related Protein p270 Is a Component of Mammalian SWI/SNF Complexes

    PubMed Central

    Dallas, Peter B.; Cheney, Ian Wayne; Liao, Da-Wei; Bowrin, Valerie; Byam, Whitney; Pacchione, Stephen; Kobayashi, Ryuji; Yaciuk, Peter; Moran, Elizabeth

    1998-01-01