Fractionation, partial characterization and bioactivity of water-soluble polysaccharides and polysaccharide-protein complexes from Pleurotus geesteranus.

PubMed

Zhang, Mei; Zhu, Lin; Cui, Steve W; Wang, Qi; Zhou, Ting; Shen, Hengsheng

2011-01-01

Fractionation and purification of mushroom polysaccharides is a critical process for mushroom clinical application. After a hot-water treatment, the crude Pleurotus geesteranus (PG) was further fractionated into four fractions (PG-1, -2, -3, -4) using gradient precipitation with water and ammonia sulphate. By controlling the initial polymer concentration and ratio of solvents, this process produced PG fractions with high chemical uniformity and narrow Mw distribution without free proteins. Structurally, PG-1 and PG-2 are pure homopolysaccharide mainly composed of glucose; and PG-3 and PG-4 are heteropolysaccharide-protein complexes. PG-2, a high M(w) fraction mainly composed of glucose presented significant cytotoxicity at the concentration of 200 and 100 μg/ml to human breast cancer cells. Here, we report a new mushroom polysaccharides extraction and fractionation method, with which we produced four fractions of PG with PG-2 appearing effective anti-tumour activity. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

The Anaerobe-Specific Orange Protein Complex of Desulfovibrio vulgaris Hildenborough Is Encoded by Two Divergent Operons Coregulated by σ54 and a Cognate Transcriptional Regulator▿†

PubMed Central

Fiévet, Anouchka; My, Laetitia; Cascales, Eric; Ansaldi, Mireille; Pauleta, Sofia R.; Moura, Isabel; Dermoun, Zorah; Bernard, Christophe S.; Dolla, Alain; Aubert, Corinne

2011-01-01

Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters DVU2103-DVU2104-DVU2105 (orp2) and DVU2107-DVU2108-DVU2109 (orp1) form two divergent operons transcribed by the σ54-RNA polymerase. We further demonstrate that the σ54-dependent transcriptional regulator DVU2106, located between orp1 and orp2, collaborates with σ54-RNA polymerase to orchestrate the simultaneous expression of the divergent orp operons. DVU2106, whose structural gene is transcribed by the σ70-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for orp operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed. PMID:21531797

Electron cryo-tomographic structure of cystovirus phi 12.

PubMed

Hu, Guo-Bin; Wei, Hui; Rice, William J; Stokes, David L; Gottlieb, Paul

2008-03-01

Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed "the donut" and appears to inhibit BHT-induced viral envelope fusion.

Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

PubMed

Liberek, K; Osipiuk, J; Zylicz, M; Ang, D; Skorko, J; Georgopoulos, C

1990-02-25

The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

Domain organizations of modular extracellular matrix proteins and their evolution.

PubMed

Engel, J

1996-11-01

Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

The nuclear retention signal of HPV16 L2 protein is essential for incoming viral genome to transverse the trans-Golgi network

PubMed Central

DiGiuseppe, Stephen; Bienkowska-Haba, Malgorzata; Hilbig, Lydia; Sapp, Martin

2014-01-01

The Human papillomavirus (HPV) capsid is composed of the major and minor capsid proteins, L1 and L2, respectively. Infectious entry requires a complex series of conformational changes in both proteins that lead to uptake and allow uncoating to occur. During entry, the capsid is disassembled and host cyclophilins dissociate L1 protein from the L2/DNA complex. Herein, we describe a mutant HPV16 L2 protein (HPV16 L2-R302/5A) that traffics pseudogenome to the trans-Golgi network (TGN) but fails to egress. Our data provide further evidence that HPV16 traffics through the TGN and demonstrates that L2 is essential for TGN egress. Furthermore, we show that cyclophilin activity is required for the L2/DNA complex to be transported to the TGN which is accompanied by a reduced L1 protein levels. PMID:24928042

Overall Architecture of the Intraflagellar Transport (IFT)-B Complex Containing Cluap1/IFT38 as an Essential Component of the IFT-B Peripheral Subcomplex.

PubMed

Katoh, Yohei; Terada, Masaya; Nishijima, Yuya; Takei, Ryota; Nozaki, Shohei; Hamada, Hiroshi; Nakayama, Kazuhisa

2016-05-20

Intraflagellar transport (IFT) is essential for assembly and maintenance of cilia and flagella as well as ciliary motility and signaling. IFT is mediated by multisubunit complexes, including IFT-A, IFT-B, and the BBSome, in concert with kinesin and dynein motors. Under high salt conditions, purified IFT-B complex dissociates into a core subcomplex composed of at least nine subunits and at least five peripherally associated proteins. Using the visible immunoprecipitation assay, which we recently developed as a convenient protein-protein interaction assay, we determined the overall architecture of the IFT-B complex, which can be divided into core and peripheral subcomplexes composed of 10 and 6 subunits, respectively. In particular, we identified TTC26/IFT56 and Cluap1/IFT38, neither of which was included with certainty in previous models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous expression of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to other IFT-B components. The detailed interaction map as well as comparison of subcellular localization of IFT-B components between wild-type and Cluap1-deficient cells provides insights into the functional relevance of the architecture of the IFT-B complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Overall Architecture of the Intraflagellar Transport (IFT)-B Complex Containing Cluap1/IFT38 as an Essential Component of the IFT-B Peripheral Subcomplex*

PubMed Central

Katoh, Yohei; Terada, Masaya; Nishijima, Yuya; Takei, Ryota; Nozaki, Shohei; Hamada, Hiroshi; Nakayama, Kazuhisa

2016-01-01

Intraflagellar transport (IFT) is essential for assembly and maintenance of cilia and flagella as well as ciliary motility and signaling. IFT is mediated by multisubunit complexes, including IFT-A, IFT-B, and the BBSome, in concert with kinesin and dynein motors. Under high salt conditions, purified IFT-B complex dissociates into a core subcomplex composed of at least nine subunits and at least five peripherally associated proteins. Using the visible immunoprecipitation assay, which we recently developed as a convenient protein-protein interaction assay, we determined the overall architecture of the IFT-B complex, which can be divided into core and peripheral subcomplexes composed of 10 and 6 subunits, respectively. In particular, we identified TTC26/IFT56 and Cluap1/IFT38, neither of which was included with certainty in previous models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous expression of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to other IFT-B components. The detailed interaction map as well as comparison of subcellular localization of IFT-B components between wild-type and Cluap1-deficient cells provides insights into the functional relevance of the architecture of the IFT-B complex. PMID:26980730

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

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

Klopffleisch, Karsten; Phan, Nguyen; Chen, Jay

2011-01-01

The heterotrimeric G-protein complex is minimally composed of G{alpha}, G{beta}, and G{gamma} subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, wemore » detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.« less

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

PubMed

Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-09-27

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

PubMed Central

Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-01-01

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification. PMID:21952135

Self-assembly in the ferritin nano-cage protein superfamily.

PubMed

Zhang, Yu; Orner, Brendan P

2011-01-01

Protein self-assembly, through specific, high affinity, and geometrically constraining protein-protein interactions, can control and lead to complex cellular nano-structures. Establishing an understanding of the underlying principles that govern protein self-assembly is not only essential to appreciate the fundamental biological functions of these structures, but could also provide a basis for their enhancement for nano-material applications. The ferritins are a superfamily of well studied proteins that self-assemble into hollow cage-like structures which are ubiquitously found in both prokaryotes and eukaryotes. Structural studies have revealed that many members of the ferritin family can self-assemble into nano-cages of two types. Maxi-ferritins form hollow spheres with octahedral symmetry composed of twenty-four monomers. Mini-ferritins, on the other hand, are tetrahedrally symmetric, hollow assemblies composed of twelve monomers. This review will focus on the structure of members of the ferritin superfamily, the mechanism of ferritin self-assembly and the structure-function relations of these proteins.

Dissecting the Calcium-Induced Differentiation of Human Primary Keratinocytes Stem Cells by Integrative and Structural Network Analyses

PubMed Central

Toufighi, Kiana; Yang, Jae-Seong; Luis, Nuno Miguel; Aznar Benitah, Salvador; Lehner, Ben; Serrano, Luis; Kiel, Christina

2015-01-01

The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55%) are composed of non-dynamic and dynamic gene products (‘di-chromatic’), 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation. PMID:25946651

Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency.

PubMed

Stein, Colleen S; Jadiya, Pooja; Zhang, Xiaoming; McLendon, Jared M; Abouassaly, Gabrielle M; Witmer, Nathan H; Anderson, Ethan J; Elrod, John W; Boudreau, Ryan L

2018-06-26

Mitochondria are composed of many small proteins that control protein synthesis, complex assembly, metabolism, and ion and reactive oxygen species (ROS) handling. We show that a skeletal muscle- and heart-enriched long non-coding RNA, LINC00116, encodes a highly conserved 56-amino-acid microprotein that we named mitoregulin (Mtln). Mtln localizes to the inner mitochondrial membrane, where it binds cardiolipin and influences protein complex assembly. In cultured cells, Mtln overexpression increases mitochondrial membrane potential, respiration rates, and Ca 2+ retention capacity while decreasing mitochondrial ROS and matrix-free Ca 2+ . Mtln-knockout mice display perturbations in mitochondrial respiratory (super)complex formation and activity, fatty acid oxidation, tricarboxylic acid (TCA) cycle enzymes, and Ca 2+ retention capacity. Blue-native gel electrophoresis revealed that Mtln co-migrates alongside several complexes, including the complex I assembly module, complex V, and supercomplexes. Under denaturing conditions, Mtln remains in high-molecular-weight complexes, supporting its role as a sticky molecular tether that enhances respiratory efficiency by bolstering protein complex assembly and/or stability. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis.

PubMed

Godlewska, Renata; Wiśniewska, Katarzyna; Pietras, Zbigniew; Jagusztyn-Krynicka, Elzbieta Katarzyna

2009-09-01

The protein Pal (peptidoglycan-associated lipoprotein) is anchored in the outer membrane (OM) of Gram-negative bacteria and interacts with Tol proteins. Tol-Pal proteins form two complexes: the first is composed of three inner membrane Tol proteins (TolA, TolQ and TolR); the second consists of the TolB and Pal proteins linked to the cell's OM. These complexes interact with one another forming a multiprotein membrane-spanning system. It has recently been demonstrated that Pal is essential for bacterial survival and pathogenesis, although its role in virulence has not been clearly defined. This review summarizes the available data concerning the structure and function of Pal and its role in pathogenesis.

Cohesin and Human Disease

PubMed Central

Liu, Jinglan; Krantz, Ian D.

2016-01-01

Cornelia de Lange syndrome (CdLS) is a dominant multisystem disorder caused by a disruption of cohesin function. The cohesin ring complex is composed of four protein subunits and more than 25 additional proteins involved in its regulation. The discovery that this complex also has a fundamental role in long-range regulation of transcription in Drosophila has shed light on the mechanism likely responsible for its role in development. In addition to the three cohesin proteins involved in CdLS, a second multisystem, recessively inherited, developmental disorder, Roberts-SC phocomelia, is caused by mutations in another regulator of the cohesin complex, ESCO2. Here we review the phenotypes of these disorders, collectively termed cohesinopathies, as well as the mechanism by which cohesin disruption likely causes these diseases. PMID:18767966

Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication

PubMed Central

Martinez, Matthew P.; Wacker, Amanda L.; Bruck, Irina; Kaplan, Daniel L.

2017-01-01

The replicative helicase unwinds parental double-stranded DNA at a replication fork to provide single-stranded DNA templates for the replicative polymerases. In eukaryotes, the replicative helicase is composed of the Cdc45 protein, the heterohexameric ring-shaped Mcm2-7 complex, and the tetrameric GINS complex (CMG). The CMG proteins bind directly to DNA, as demonstrated by experiments with purified proteins. The mechanism and function of these DNA-protein interactions are presently being investigated, and a number of important discoveries relating to how the helicase proteins interact with DNA have been reported recently. While some of the protein-DNA interactions directly relate to the unwinding function of the enzyme complex, other protein-DNA interactions may be important for minichromosome maintenance (MCM) loading, origin melting or replication stress. This review describes our current understanding of how the eukaryotic replicative helicase subunits interact with DNA structures in vitro, and proposed models for the in vivo functions of replicative helicase-DNA interactions are also described. PMID:28383499

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reagent Anions for Charge Inversion of Polypeptide/Protein Cations in the Gas Phase

PubMed Central

He, Min; Emory, Joshua F.; McLuckey, Scott A.

2005-01-01

Various reagent anions capable of converting polypeptide cations to anions via ion/ion reactions have been investigated. The major charge inversion reaction channels include multiple proton transfer and adduct formation. Dianions composed of sulfonate groups as the negative charge carriers show essentially exclusive adduct formation in converting protonated peptides and proteins to anions. Dianions composed of carboxylate groups, on the other hand, show far more charge inversion via multiple proton transfer, with the degree of adduct formation dependent upon both the size of the polypeptide and the spacings between carboxylate groups in the dianion. More highly charged carboxylate-containing anions, such as those derived from carboxylate-terminated polyamidoamine half-generation dendrimers show charge inversion to give anion charges as high in magnitude as −4, with the degree of adduct formation being inversely related to dendrimer generation. All observations can be interpreted on the basis of charge inversion taking place via a long-lived chemical complex. The lifetime of this complex is related to the strengths and numbers of the interactions of the reactants in the complex. Calculations with model systems are fully consistent with sulfonate groups giving rise to more stable complexes. The kinetic stability of the complex can also be affected by the presence of electrostatic repulsion if it is multiply charged. In general, this situation destabilizes the complex and reduces the likelihood for observation of adducts. The findings highlight the characteristics of multiply charged anions that play roles in determining the nature of charge inversion products associated with protonated peptides and proteins. PMID:15889906

Ontogenetical and histochemical studies on the translator apparatus in Calotropis procera R. Br. I. The retinaculum.

PubMed

Vijayaraghavan, M R; Cheema, K

1977-01-01

Translator apparatus consists of 2 parts--the corpusculum, which is attached to the margin of the stigmatic head between the anthers, and a pair of arms by which the corpusculum is attached to the pollinia of the adjacent anther halves. Retinaculum spans the lateral blade of the corpusculum to the acellular beak of the pollinium. Ontogenetically, it is the secretory product of the stigma along the secondary stigmatic groove. Histochemically, it is composed of lipid, lignin, cutin and a little amount of proteins. Retinaculum, like corpusculum, is stigmatic in origin, but differs histochemically from the latter being composed mainly of lipo-protein complex and lacking phenolic compounds.

Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

PubMed

Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki

2015-06-01

Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The CmrΔ1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 Å, α = 90.3, β = 104.8, γ = 118.6°. The asymmetric unit of the crystals is expected to contain one CmrΔ1-ssDNA complex, with a Matthews coefficient of 2.03 Å(3) Da(-1) and a solvent content of 39.5%.

Expression, Refolding and Crystallizations of the Grb2-like (GADS) C-Terminal SH3 Domain Complexed with a SLP-76 Motif Peptide

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

Faravelli,A.; Dimasi, N.

The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.

Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

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

Faravelli, Alessandro; Dimasi, Nazzareno, E-mail: ndimasi@gmail.com

Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli,more » refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.« less

LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins

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

Sosa, Brian A.; Rothballer, Andrea; Kutay, Ulrike

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansivemore » grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.« less

Anticoagulant activity of marine green and brown algae collected from Jeju Island in Korea.

PubMed

Athukorala, Yasantha; Lee, Ki-Wan; Kim, Se-Kwon; Jeon, You-Jin

2007-07-01

Twenty-two algal species were evaluated for their potential anticoagulant activities. Hot water extracts from selected species, Codium fragile and Sargassum horneri showed high activated partial thromboplastin time (APTT). Ultraflo extract of C. fragile and S. horneri exhibited the most potent anticoagulant activity. Furthermore, in both algal species, active compounds were mainly concentrated in >30kDa faction. The crude polysaccharide fraction (>30kDa; CpoF) of C. fragile composed of approximately 80% carbohydrate and approximately 19% of protein; the crude polysaccharide fraction (>30kDa; CpoF) of S. horneri was composed of 97% of carbohydrate and approximately 2% of protein. Therefore, most probably the active compound, or compounds of the algal species were related to high molecular weight polysaccharide, or a complex form with carbohydrate and protein (proteoglycan).

Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement.

PubMed

Kublik, Anja; Deobald, Darja; Hartwig, Stefanie; Schiffmann, Christian L; Andrades, Adarelys; von Bergen, Martin; Sawers, R Gary; Adrian, Lorenz

2016-09-01

Dehalococcoides mccartyi strain CBDB1 is an obligate organohalide-respiring bacterium using only hydrogen as electron donor and halogenated organics as electron acceptor. Here, we studied proteins involved in the respiratory chain under non-denaturing conditions. Using blue native gel electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250-270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster containing subunit (CbdbA131) of the hydrogen uptake hydrogenase (Hup). No colocalisation between the catalytically active subunits of hydrogenase and reductive dehalogenase was found. By two-dimensional BN/SDS-PAGE the stability of the complex towards detergents was assessed, demonstrating stepwise disintegration with increasing detergent concentrations. Chemical cross-linking confirmed the presence of a higher molecular mass reductive dehalogenase protein complex composed of RdhA, CISM I and Hup hydrogenase and proved to be a potential tool for stabilising protein-protein interactions of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics

PubMed Central

Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

2016-01-01

The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics. PMID:26732734

Purification and characterization of a cellulolytic multienzyme complex produced by Neocallimastix patriciarum J11.

PubMed

Wang, Hui-Chang; Chen, Yo-Chia; Hseu, Ruey-Shyang

2014-08-22

Understanding the roles of the components of the multienzyme complex of the anaerobial cellulase system, acting on complex substrates, is crucial to the development of efficient cellulase systems for industrial applications such as converting lignocellulose to sugars for bioethanol production. In this study, we purified the multienzyme complex of Neocallimastix patriciarum J11 from a broth through cellulose affinity purification. The multienzyme complex is composed of at least 12 comprised proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eight of these constituents have demonstrated β-glucanase activity on zymogram analysis. The multienzyme complex contained scaffoldings that respond to the gathering of the cellulolytic components. The levels and subunit ratio of the multienzyme complex from N. patriciarum J11 might have been affected by their utilized carbon sources, whereas the components of the complexes were consistent. The trypsin-digested peptides of six proteins were matched to the sequences of cellulases originating from rumen fungi, based on identification through liquid chromatography/mass spectrometry, revealing that at least three types of cellulase, including one endoglucanase and two exoglucanases, could be found in the multienzyme complex of N. patriciarum J11. The cellulolytic subunits could hydrolyze synergistically on both the internal bonds and the reducing and nonreducing ends of cellulose. Based on our research, our findings are the first to depict the composition of the multienzyme complex produced by N. patriciarum J11, and this complex is composed of scaffoldin and three types of cellulase. Copyright © 2014 Elsevier Inc. All rights reserved.

Chemical cross-linking of the urease complex from Helicobacter pylori and analysis by Fourier transform ion cyclotron resonance mass spectrometry and molecular modeling

NASA Astrophysics Data System (ADS)

Carlsohn, Elisabet; Ångström, Jonas; Emmett, Mark R.; Marshall, Alan G.; Nilsson, Carol L.

2004-05-01

Chemical cross-linking of proteins is a well-established method for structural mapping of small protein complexes. When combined with mass spectrometry, cross-linking can reveal protein topology and identify contact sites between the peptide surfaces. When applied to surface-exposed proteins from pathogenic organisms, the method can reveal structural details that are useful in vaccine design. In order to investigate the possibilities of applying cross-linking on larger protein complexes, we selected the urease enzyme from Helicobacter pylori as a model. This membrane-associated protein complex consists of two subunits: [alpha] (26.5 kDa) and [beta] (61.7 kDa). Three ([alpha][beta]) heterodimers form a trimeric ([alpha][beta])3 assembly which further associates into a unique dodecameric 1.1 MDa complex composed of four ([alpha][beta])3 units. Cross-linked peptides from trypsin-digested urease complex were analyzed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and molecular modeling. Two potential cross-linked peptides (present in the cross-linked sample but undetectable in [alpha], [beta], and native complex) were assigned. Molecular modeling of urease [alpha][beta] complex and trimeric urease units ([alpha][beta])3 revealed a linkage site between the [alpha]-subunit and the [beta]-subunit, and an internal cross-linkage in the [beta]-subunit.

Cellular and synaptic network defects in autism

PubMed Central

Peça, João; Feng, Guoping

2012-01-01

Many candidate genes are now thought to confer susceptibility to autism spectrum disorder (ASD). Here we review four interrelated complexes, each composed of multiple families of genes that functionally coalesce on common cellular pathways. We illustrate a common thread in the organization of glutamatergic synapses and suggest a link between genes involved in Tuberous Sclerosis Complex, Fragile X syndrome, Angelman syndrome and several synaptic ASD candidate genes. When viewed in this context, progress in deciphering the molecular architecture of cellular protein-protein interactions together with the unraveling of synaptic dysfunction in neural networks may prove pivotal to advancing our understanding of ASDs. PMID:22440525

Single-molecule fluorescence measurements reveal the reaction mechanisms of the core RISC, composed of human Argonaute 2 and a guide RNA.

PubMed

Jo, Myung Hyun; Song, Ji-Joon; Hohng, Sungchul

2015-12-01

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection.

Blood coagulation reactions on nanoscale membrane surfaces

NASA Astrophysics Data System (ADS)

Pureza, Vincent S.

Blood coagulation requires the assembly of several membrane-bound protein complexes composed of regulatory and catalytic subunits. The biomembranes involved in these reactions not only provide a platform for these procoagulant proteins, but can also affect their function. Increased exposure of acidic phospholipids on the outer leaflet of the plasma membrane can dramatically modulate the catalytic efficiencies of such membrane-bound enzymes. Under physiologic conditions, however, these phospholipids spontaneously cluster into a patchwork of membrane microdomains upon which membrane binding proteins may preferentially assemble. As a result, the membrane composition surrounding these proteins is largely unknown. Through the development and use of a nanometer-scale bilayer system that provides rigorous control of the phospholipid membrane environment, I investigated the role of phosphatidylserine, an acidic phospholipid, in the direct vicinity (within nanometers) of two critical membrane-bound procoagulant protein complexes and their respective natural substrates. Here, I present how the assembly and function of the tissue factor˙factor VIIa and factor Va˙factor Xa complexes, the first and final cofactor˙enzyme complexes of the blood clotting cascade, respectively, are mediated by changes in their immediate phospholipid environments.

Proteomic analysis in non-denaturing condition of the secretome reveals the presence of multienzyme complexes in Penicillium purpurogenum.

PubMed

Gonzalez-Vogel, Alvaro; Eyzaguirre, Jaime; Oleas, Gabriela; Callegari, Eduardo; Navarrete, Mario

2011-01-01

Proteins secreted by filamentous fungi play key roles in different aspects of their biology. The fungus Penicillium purpurogenum, used as a model organism, is able to degrade hemicelluloses and pectins by secreting a variety of enzymes to the culture medium. This work shows that these enzymes interact with each other to form high molecular weight, catalytically active complexes. By using a proteomics approach, we were able to identify several protein complexes in the secretome of this fungus. The expression and assembly of these complexes depend on the carbon source used and display molecular masses ranging from 300 to 700 kDa. These complexes are composed of a variety of enzymes, including arabinofuranosidases, acetyl xylan esterases, feruloyl esterases, β-glucosidases and xylanases. The protein-protein interactions in these multienzyme complexes were confirmed by coimmunoprecipitation assays. One of the complexes was purified from sugar beet pulp cultures and the subunits identified by tandem mass spectrometry. A better understanding of the biological significance of these kinds of interactions will help in the comprehension of the degradation mechanisms used by fungi and may be of special interest to the biotechnology industry.

Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1

PubMed Central

Klockenbusch, Cordula; Kast, Juergen

2010-01-01

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively. PMID:20634879

Regulation of mTORC1 by PI3K signaling.

PubMed

Dibble, Christian C; Cantley, Lewis C

2015-09-01

The class I phosphoinositide 3-kinase (PI3K)-mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling network directs cellular metabolism and growth. Activation of mTORC1 [composed of mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal with SEC13 protein 8(mLST8), 40-kDa proline-rich Akt substrate (PRAS40), and DEP domain-containing mTOR-interacting protein (DEPTOR)] depends on the Ras-related GTPases (Rags) and Ras homolog enriched in brain (Rheb) GTPase and requires signals from amino acids, glucose, oxygen, energy (ATP), and growth factors (including cytokines and hormones such as insulin). Here we discuss the signal transduction mechanisms through which growth factor-responsive PI3K signaling activates mTORC1. We focus on how PI3K-dependent activation of Akt and spatial regulation of the tuberous sclerosis complex (TSC) complex (TSC complex) [composed of TSC1, TSC2, and Tre2-Bub2-Cdc16-1 domain family member 7 (TBC1D7)] switches on Rheb at the lysosome, where mTORC1 is activated. Integration of PI3K- and amino acid-dependent signals upstream of mTORC1 at the lysosome is detailed in a working model. A coherent understanding of the PI3K-mTORC1 network is imperative as its dysregulation has been implicated in diverse pathologies including cancer, diabetes, autism, and aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

The CWB2 Cell Wall-Anchoring Module Is Revealed by the Crystal Structures of the Clostridium difficile Cell Wall Proteins Cwp8 and Cwp6.

PubMed

Usenik, Aleksandra; Renko, Miha; Mihelič, Marko; Lindič, Nataša; Borišek, Jure; Perdih, Andrej; Pretnar, Gregor; Müller, Uwe; Turk, Dušan

2017-03-07

Bacterial cell wall proteins play crucial roles in cell survival, growth, and environmental interactions. In Gram-positive bacteria, cell wall proteins include several types that are non-covalently attached via cell wall binding domains. Of the two conserved surface-layer (S-layer)-anchoring modules composed of three tandem SLH or CWB2 domains, the latter have so far eluded structural insight. The crystal structures of Cwp8 and Cwp6 reveal multi-domain proteins, each containing an embedded CWB2 module. It consists of a triangular trimer of Rossmann-fold CWB2 domains, a feature common to 29 cell wall proteins in Clostridium difficile 630. The structural basis of the intact module fold necessary for its binding to the cell wall is revealed. A comparison with previously reported atomic force microscopy data of S-layers suggests that C. difficile S-layers are complex oligomeric structures, likely composed of several different proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Assembly of the Human Signal Recognition Particle

NASA Astrophysics Data System (ADS)

Menichelli, Elena; Nagai, Kiyoshi

Large RNA-protein complexes (ribonucleoprotein particles or RNPs) control fundamental biological processes. Their correct assembly is essential for function and occurs by the ordered addition of proteins to the RNA. A good model system for studying RNP assembly is provided by the Signal Recognition Particle (SRP), an RNP conserved from bacteria to humans, with different degrees of complexity. Human SRP, composed of a single RNA molecule and six pro teins, is responsible for the co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum membrane. In vitro studies reveal that the SRP proteins need to be added to the RNA sequentially. If the order of addition is altered, non-native particles are formed. The sequential association of proteins causes conformational changes in the RNA, allowing binding of other proteins. The in vivo assembly is regulated by the translocation of precursors between different cellular compartments. In this chapter we review the current understanding of the human SRP assembly mechanism.

Quantitative Interpretation of Multifrequency Multimode EPR Spectra of Metal Containing Proteins, Enzymes, and Biomimetic Complexes.

PubMed

Petasis, Doros T; Hendrich, Michael P

2015-01-01

Electron paramagnetic resonance (EPR) spectroscopy has long been a primary method for characterization of paramagnetic centers in materials and biological complexes. Transition metals in biological complexes have valence d-orbitals that largely define the chemistry of the metal centers. EPR spectra are distinctive for metal type, oxidation state, protein environment, substrates, and inhibitors. The study of many metal centers in proteins, enzymes, and biomimetic complexes has led to the development of a systematic methodology for quantitative interpretation of EPR spectra from a wide array of metal containing complexes. The methodology is now contained in the computer program SpinCount. SpinCount allows simulation of EPR spectra from any sample containing multiple species composed of one or two metals in any spin state. The simulations are quantitative, thus allowing determination of all species concentrations in a sample directly from spectra. This chapter will focus on applications to transition metals in biological systems using EPR spectra from multiple microwave frequencies and modes. © 2015 Elsevier Inc. All rights reserved.

PsB multiprotein complex of Dictyostelium discoideum. Demonstration of cellulose binding activity and order of protein subunit assembly.

PubMed

McGuire, V; Alexander, S

1996-06-14

The differentiated spores of Dictyostelium are surrounded by an extracellular matrix, the spore coat, which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique extracellular matrix is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, N., McGuire, V., and Alexander, S (1994) J. Cell Sci. 107, 2567-2579). Later in development, the complex is synchronously secreted from the prespore vesicles and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat.

Characterization, stoichiometry, and stability of salivary protein-tannin complexes by ESI-MS and ESI-MS/MS.

PubMed

Canon, Francis; Paté, Franck; Meudec, Emmanuelle; Marlin, Thérèse; Cheynier, Véronique; Giuliani, Alexandre; Sarni-Manchado, Pascale

2009-12-01

Numerous protein-polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP-tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP-tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5.EgCG complexes are maintained intact in the gas phase.

The structural basis of transferrin sequestration by transferrin-binding protein B

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

Calmettes, Charles; Alcantara, Joenel; Yu, Rong-Hua

2012-03-28

Neisseria meningitidis, the causative agent of bacterial meningitis, acquires the essential element iron from the host glycoprotein transferrin during infection through a surface transferrin receptor system composed of proteins TbpA and TbpB. Here we present the crystal structures of TbpB from N. meningitidis in its apo form and in complex with human transferrin. The structure reveals how TbpB sequesters and initiates iron release from human transferrin.

Effects of novel adjuvant complex/Eimeria profilin vaccine on intestinal host immune responses against live E. acervulina challenge infection

USDA-ARS?s Scientific Manuscript database

The effects of a novel adjuvant; composed of Quil A, cholesterol, dimethyl dioctadecyl ammonium bromide, and Carbopol (QCDC), on protective immunity against avian coccidiosis following immunization with an Eimeria recombinant protein were determined. Broiler chickens were subcutaneously immunized w...

Chemical tools to investigate mechanisms associated with HSP90 and HSP70 in disease

PubMed Central

Shrestha, Liza; Patel, Hardik J.; Chiosis, Gabriela

2016-01-01

The chaperome is a large and diverse protein machinery composed of chaperone proteins and a variety of helpers, such as the co-chaperones, folding enzymes and scaffolding and adapter proteins. Heat shock protein 90s and 70s (HSP90s and HSP70s), the most abundant chaperome members in human cells, are also the most complex. As we have learned to appreciate, their functions are context dependent and manifested through a variety of conformations that each recruit a subset of co-chaperone, scaffolding and folding proteins and which are further diversified by the post-translational modifications each carry, making their study through classic genetic and biochemical techniques quite a challenge. Chemical biology tools and techniques have been developed over the years to help decipher the complexities of the HSPs and this review will provide an overview of such efforts with focus on HSP90 and HSP70. PMID:26933742

Purification and partial characterization of a lectin protein complex, the clathrilectin, from the calcareous sponge Clathrina clathrus.

PubMed

Gardères, Johan; Domart-Coulon, Isabelle; Marie, Arul; Hamer, Bojan; Batel, Renato; Müller, Werner E G; Bourguet-Kondracki, Marie-Lise

2016-10-01

Carbohydrate-binding proteins were purified from the marine calcareous sponge Clathrina clathrus via affinity chromatography on lactose and N-acetyl glucosamine-agarose resins. Proteomic analysis of acrylamide gel separated protein subunits obtained in reducing conditions pointed out several candidates for lectins. Based on amino-acid sequence similarity, two peptides displayed homology with the jack bean lectin Concanavalin A, including a conserved domain shared by proteins in the L-type lectin superfamily. An N-acetyl glucosamine - binding protein complex, named clathrilectin, was further purified via gel filtration chromatography, bioguided with a diagnostic rabbit erythrocyte haemagglutination assay, and its activity was found to be calcium dependent. Clathrilectin, a protein complex of 3200kDa estimated by gel filtration, is composed of monomers with apparent molecular masses of 208 and 180kDa estimated on 10% SDS-PAGE. Nine internal peptides were identified using proteomic analyses, and compared to protein libraries from the demosponge Amphimedon queenslandica and a calcareous sponge Sycon sp. from the Adriatic Sea. The clathrilectin is the first lectin isolated from a calcareous sponge and displays homologies with predicted sponge proteins potentially involved in cell aggregation and interaction with bacteria. Copyright © 2016 Elsevier Inc. All rights reserved.

Prioritisation of associations between protein domains and complex diseases using domain-domain interaction networks.

PubMed

Wang, W; Zhang, W; Jiang, R; Luan, Y

2010-05-01

It is of vital importance to find genetic variants that underlie human complex diseases and locate genes that are responsible for these diseases. Since proteins are typically composed of several structural domains, it is reasonable to assume that harmful genetic variants may alter structures of protein domains, affect functions of proteins and eventually cause disorders. With this understanding, the authors explore the possibility of recovering associations between protein domains and complex diseases. The authors define associations between protein domains and disease families on the basis of associations between non-synonymous single nucleotide polymorphisms (nsSNPs) and complex diseases, similarities between diseases, and relations between proteins and domains. Based on a domain-domain interaction network, the authors propose a 'guilt-by-proximity' principle to rank candidate domains according to their average distance to a set of seed domains in the domain-domain interaction network. The authors validate the method through large-scale cross-validation experiments on simulated linkage intervals, random controls and the whole genome. Results show that areas under receiver operating characteristic curves (AUC scores) can be as high as 77.90%, and the mean rank ratios can be as low as 21.82%. The authors further offer a freely accessible web interface for a genome-wide landscape of associations between domains and disease families.

Meiosis in male Drosophila

PubMed Central

McKee, Bruce D.; Yan, Rihui; Tsai, Jui-He

2012-01-01

Meiosis entails sorting and separating both homologous and sister chromatids. The mechanisms for connecting sister chromatids and homologs during meiosis are highly conserved and include specialized forms of the cohesin complex and a tightly regulated homolog synapsis/recombination pathway designed to yield regular crossovers between homologous chromatids. Drosophila male meiosis is of special interest because it dispenses with large segments of the standard meiotic script, particularly recombination, synapsis and the associated structures. Instead, Drosophila relies on a unique protein complex composed of at least two novel proteins, SNM and MNM, to provide stable connections between homologs during meiosis I. Sister chromatid cohesion in Drosophila is mediated by cohesins, ring-shaped complexes that entrap sister chromatids. However, unlike other eukaryotes Drosophila does not rely on the highly conserved Rec8 cohesin in meiosis, but instead utilizes two novel cohesion proteins, ORD and SOLO, which interact with the SMC1/3 cohesin components in providing meiotic cohesion. PMID:23087836

Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

NASA Astrophysics Data System (ADS)

Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

2011-07-01

Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

Biological and Pathological Implications of an Alternative ATP-Powered Proteasomal Assembly With Cdc48 and the 20S Peptidase.

PubMed

Esaki, Masatoshi; Johjima-Murata, Ai; Islam, Md Tanvir; Ogura, Teru

2018-01-01

The ATP-powered protein degradation machinery plays essential roles in maintaining protein homeostasis in all organisms. Robust proteolytic activities are typically sequestered within protein complexes to avoid the fatal removal of essential proteins. Because the openings of proteolytic chambers are narrow, substrate proteins must undergo unfolding. AAA superfamily proteins (ATPases associated with diverse cellular activities) are mostly located at these openings and regulate protein degradation appropriately. The 26S proteasome, comprising 20S peptidase and 19S regulatory particles, is the major ATP-powered protein degradation machinery in eukaryotes. The 19S particles are composed of six AAA proteins and 13 regulatory proteins, and bind to both ends of a barrel-shaped proteolytic chamber formed by the 20S peptidase. Several recent studies have reported that another AAA protein, Cdc48, can replace the 19S particles to form an alternative ATP-powered proteasomal complex, i.e., the Cdc48-20S proteasome. This review focuses on our current knowledge of this alternative proteasome and its possible linkage to amyotrophic lateral sclerosis.

The Alternative complex III: properties and possible mechanisms for electron transfer and energy conservation.

PubMed

Refojo, Patrícia N; Teixeira, Miguel; Pereira, Manuela M

2012-10-01

Alternative complexes III (ACIII) are recently identified membrane-bound enzymes that replace functionally the cytochrome bc(1/)b(6)f complexes. In general, ACIII are composed of four transmembrane proteins and three peripheral subunits that contain iron-sulfur centers and C-type hemes. ACIII are built by a combination of modules present in different enzyme families, namely the complex iron-sulfur molybdenum containing enzymes. In this article a historical perspective on the investigation of ACIII is presented, followed by an overview of the present knowledge on these enzymes. Electron transfer pathways within the protein are discussed taking into account possible different locations (cytoplasmatic or periplasmatic) of the iron-sulfur containing protein and their contribution to energy conservation. In this way several hypotheses for energy conservation modes are raised including linear and bifurcating electron transfer pathways. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). Copyright © 2012 Elsevier B.V. All rights reserved.

Retriever, a multiprotein complex for retromer-independent endosomal cargo recycling

PubMed Central

McNally, Kerrie E.; Faulkner, Rebecca; Steinberg, Florian; Gallon, Matthew; Ghai, Rajesh; Pim, David; Langton, Paul; Pearson, Neil; Danson, Chris M.; Nägele, Heike; Morris, Lindsey M; Singla, Arnika; Overlee, Brittany L; Heesom, Kate J.; Sessions, Richard; Banks, Lawrence; Collins, Brett M; Berger, Imre; Billadeau, Daniel D.; Burstein, Ezra; Cullen, Peter J.

2018-01-01

Following endocytosis and entry into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are alternatively retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multi-protein complex which orchestrates cargo retrieval and recycling and importantly, is biochemically and functionally distinct to the established retromer pathway. Composed of a heterotrimer of DSCR3, C16orf62 and VPS29, and bearing striking similarity with retromer, we have called this complex ‘retriever’. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to the CCC and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5β1-integrin. Through quantitative proteomic analysis we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, which require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major new endosomal retrieval and recycling pathway. PMID:28892079

Coexisting properties of thermostability and ultraviolet radiation resistance in the main S-layer complex of Deinococcus radiodurans.

PubMed

Farci, Domenica; Slavov, Chavdar; Piano, Dario

2018-01-17

Deinococcus radiodurans is well known for its unusual resistance to different environmental stresses. Recently, we have described a novel complex composed of the surface (S)-layer protein DR_2577 and the carotenoid deinoxanthin. We also showed a role of this complex in the UV resistance under desiccation. Both these properties, UV and desiccation resistance, suggest a selective pressure generated by Sun irradiation. In order to confirm this hypothesis we checked whether this S-layer Deinoxanthin Binding Complex (SDBC) has features of thermo-resistance, a property also expected in proteins evolved under solar irradiative pressure. We performed the spectroscopic characterization of the SDBC by means of thermal shift assay, circular dichroism and related in silico analysis. Our findings identify a stability typical of thermo-adapted proteins and provide a new insight into the origin of specific S-layer types. The results are discussed in terms of co-evolutionary mechanisms related to Sun-induced desiccation and heat.

Topological properties of complex networks in protein structures

NASA Astrophysics Data System (ADS)

Kim, Kyungsik; Jung, Jae-Won; Min, Seungsik

2014-03-01

We study topological properties of networks in structural classification of proteins. We model the native-state protein structure as a network made of its constituent amino-acids and their interactions. We treat four structural classes of proteins composed predominantly of α helices and β sheets and consider several proteins from each of these classes whose sizes range from amino acids of the Protein Data Bank. Particularly, we simulate and analyze the network metrics such as the mean degree, the probability distribution of degree, the clustering coefficient, the characteristic path length, the local efficiency, and the cost. This work was supported by the KMAR and DP under Grant WISE project (153-3100-3133-302-350).

The length but not the sequence of peptide linker modules exerts the primary influence on the conformations of protein domains in cellulosome multi-enzyme complexes.

PubMed

Różycki, Bartosz; Cazade, Pierre-André; O'Mahony, Shane; Thompson, Damien; Cieplak, Marek

2017-08-16

Cellulosomes are large multi-protein catalysts produced by various anaerobic microorganisms to efficiently degrade plant cell-wall polysaccharides down into simple sugars. X-ray and physicochemical structural characterisations show that cellulosomes are composed of numerous protein domains that are connected by unstructured polypeptide segments, yet the properties and possible roles of these 'linker' peptides are largely unknown. We have performed coarse-grained and all-atom molecular dynamics computer simulations of a number of cellulosomal linkers of different lengths and compositions. Our data demonstrates that the effective stiffness of the linker peptides, as quantified by the equilibrium fluctuations in the end-to-end distances, depends primarily on the length of the linker and less so on the specific amino acid sequence. The presence of excluded volume - provided by the domains that are connected - dampens the motion of the linker residues and reduces the effective stiffness of the linkers. Simultaneously, the presence of the linkers alters the conformations of the protein domains that are connected. We demonstrate that short, stiff linkers induce significant rearrangements in the folded domains of the mini-cellulosome composed of endoglucanase Cel8A in complex with scaffoldin ScafT (Cel8A-ScafT) of Clostridium thermocellum as well as in a two-cohesin system derived from the scaffoldin ScaB of Acetivibrio cellulolyticus. We give experimentally testable predictions on structural changes in protein domains that depend on the length of linkers.

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

PubMed

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

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

Supramolecular Ferric Porphyrins as Cyanide Receptors in Aqueous Solution

PubMed Central

2011-01-01

All fundamental data about binding of the cyanide to a supramolecular complex composed of a per-O-methylated β-cyclodextrin dimer having an imidazole linker (Im3CD) and an anionic ferric porphyrin (Fe(III)TPPS) indicate that the Fe(III)TPPS/Im3CD complex is much better as an cyanide receptor in vivo than hydroxocobalamin, whose cyanide binding ability is lowered by its strong binding to serum proteins in the blood. PMID:24900285

The TOC complex: preprotein gateway to the chloroplast.

PubMed

Andrès, Charles; Agne, Birgit; Kessler, Felix

2010-06-01

Photosynthetic eukaryotes strongly depend on chloroplast metabolic pathways. Most if not all involve nuclear encoded proteins. These are synthesized as cytosolic preproteins with N-terminal, cleavable targeting sequences (transit peptide). Preproteins are imported by a major pathway composed of two proteins complexes: TOC and TIC (Translocon of the Outer and Inner membranes of the Chloroplasts, respectively). These selectively recognize the preproteins and facilitate their transport across the chloroplast envelope. The TOC core complex consists of three types of components, each belonging to a small family: Toc34, Toc75 and Toc159. Toc34 and Toc159 isoforms represent a subfamily of the GTPase superfamily. The members of the Toc34 and Toc159 subfamily act as GTP-dependent receptors at the chloroplast surface and distinct members of each occur in defined, substrate-specific TOC complexes. Toc75, a member of the Omp85 family, is conserved from prokaryotes and functions as the unique protein-conducting channel at the outer membrane. In this review we will describe the current state of knowledge regarding the composition and function of the TOC complex.

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae*

PubMed Central

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.; Shirasaki, Dyna I.; Wang, Charles; Blaby-Haas, Crysten E.; Merchant, Sabeeha S.; Loo, Joseph A.; Clarke, Catherine F.

2015-01-01

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. Here, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11. PMID:25631044

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae

DOE PAGES

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.; ...

2015-01-28

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. In thismore » paper, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q 6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Finally, given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.« less

The roles of cohesins in mitosis, meiosis, and human health and disease

PubMed Central

Brooker, Amanda S.; Berkowitz, Karen M.

2015-01-01

Summary Mitosis and meiosis are essential processes that occur during development. Throughout these processes, cohesion is required to keep the sister chromatids together until their separation at anaphase. Cohesion is created by multi-protein subunit complexes called cohesins. Although the subunits differ slightly in mitosis and meiosis, the canonical cohesin complex is composed of four subunits that are quite diverse. The cohesin complexes are also important for DNA repair, gene expression, development, and genome integrity. Here we provide an overview of the roles of cohesins during these different events, as well as their roles in human health and disease, including the cohesinopathies. Although the exact roles and mechanisms of these proteins are still being elucidated, this review will serve as a guide for the current knowledge of cohesins. PMID:24906316

The Role of Hexon Protein as a Molecular Mold in Patterning the Protein IX Organization in Human Adenoviruses.

PubMed

Reddy, Vijay S

2017-09-01

Adenoviruses are respiratory, ocular and enteric pathogens that form complex capsids, which are assembled from seven different structural proteins and composed of several core proteins that closely interact with the packaged dsDNA genome. The recent near-atomic resolution structures revealed that the interlacing continuous hexagonal network formed by the protein IX molecules is conserved among different human adenoviruses (HAdVs), but not in non-HAdVs. In this report, we propose a distinct role for the hexon protein as a "molecular mold" in enabling the formation of such hexagonal protein IX network that has been shown to preserve the stability and infectivity of HAdVs. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. In thismore » paper, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q 6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Finally, given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.« less

Molecular interactions of orthologues of floral homeotic proteins from the gymnosperm Gnetum gnemon provide a clue to the evolutionary origin of 'floral quartets'.

PubMed

Wang, Yong-Qiang; Melzer, Rainer; Theissen, Günter

2010-10-01

Several lines of evidence suggest that the identity of floral organs in angiosperms is specified by multimeric transcription factor complexes composed of MADS-domain proteins. These bind to specific cis-regulatory elements ('CArG-boxes') of their target genes involving DNA-loop formation, thus constituting 'floral quartets'. Gymnosperms, angiosperms' closest relatives, contain orthologues of floral homeotic genes, but when and how the interactions constituting floral quartets were established during evolution has remained unknown. We have comprehensively studied the dimerization and DNA-binding of several classes of MADS-domain proteins from the gymnosperm Gnetum gnemon. Determination of protein-protein and protein-DNA interactions by yeast two-hybrid, in vitro pull-down and electrophoretic mobility shift assays revealed complex patterns of homo- and heterodimerization among orthologues of floral homeotic class B, class C and class E proteins and B(sister) proteins. Using DNase I footprint assays we demonstrate that both orthologues of class B with C proteins, and orthologues of class C proteins alone, but not orthologues of class B proteins alone can loop DNA in floral quartet-like complexes. This is in contrast to class B and class C proteins from angiosperms, which require other factors such as class E floral homeotic proteins to 'glue' them together in multimeric complexes. Our findings suggest that the evolutionary origin of floral quartet formation is based on the interaction of different DNA-bound homodimers, does not depend on class E proteins, and predates the origin of angiosperms. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

Tripartite assembly of RND multidrug efflux pumps

NASA Astrophysics Data System (ADS)

Daury, Laetitia; Orange, François; Taveau, Jean-Christophe; Verchère, Alice; Monlezun, Laura; Gounou, Céline; Marreddy, Ravi K. R.; Picard, Martin; Broutin, Isabelle; Pos, Klaas M.; Lambert, Olivier

2016-02-01

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

Evolution of an RNP assembly system: A minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster

PubMed Central

Kroiss, Matthias; Schultz, Jörg; Wiesner, Julia; Chari, Ashwin; Sickmann, Albert; Fischer, Utz

2008-01-01

In vertebrates, assembly of spliceosomal uridine-rich small nuclear ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a macromolecular entity composed of the proteins SMN and Gemins 2–8. Here we have studied the evolution of this machinery using complete genome assemblies of multiple model organisms. The SMN complex has gained complexity in evolution by a blockwise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a previously undescribed biochemical purification strategy elucidated that the dipteran Drosophila melanogaster contains an SMN complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents misassembly onto nontarget RNAs. Our data suggest that only a minority of Gemins are required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. The evolution of the SMN complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction. PMID:18621711

Cell wall proteome analysis of Arabidopsis thaliana mature stems.

PubMed

Duruflé, Harold; Clemente, Hélène San; Balliau, Thierry; Zivy, Michel; Dunand, Christophe; Jamet, Elisabeth

2017-04-01

Plant stems carry flowers necessary for species propagation and need to be adapted to mechanical disturbance and environmental factors. The stem cell walls are different from other organs and can modify their rigidity or viscoelastic properties for the integrity and the robustness required to withstand mechanical impacts and environmental stresses. Plant cell wall is composed of complex polysaccharide networks also containing cell wall proteins (CWPs) crucial to perceive and limit the environmental effects. The CWPs are fundamental players in cell wall remodeling processes, and today, only 86 have been identified from the mature stems of the model plant Arabidopsis thaliana. With a destructive method, this study has enlarged its coverage to 302 CWPs. This new proteome is mainly composed of 27.5% proteins acting on polysaccharides, 16% proteases, 11.6% oxido-reductases, 11% possibly related to lipid metabolism and 11% of proteins with interacting domains with proteins or polysaccharides. Compared to stem cell wall proteomes already available (Brachypodium distachyon, Sacharum officinarum, Linum usitatissimum, Medicago sativa), that of A. thaliana stems has a higher proportion of proteins acting on polysaccharides and of proteases, but a lower proportion of oxido-reductases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity

NASA Astrophysics Data System (ADS)

Simon, Joseph R.; Carroll, Nick J.; Rubinstein, Michael; Chilkoti, Ashutosh; López, Gabriel P.

2017-06-01

Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments. Using theoretical analyses, we interpret the phase behaviour of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-separated granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-molecular-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biology and (3) molecular-level engineering of self-assembled recombinant IDP-rich materials.

Molecular mechanisms of the cytotoxicity of human α-lactalbumin made lethal to tumor cells (HAMLET) and other protein-oleic acid complexes.

PubMed

Nakamura, Takashi; Aizawa, Tomoyasu; Kariya, Ryusho; Okada, Seiji; Demura, Makoto; Kawano, Keiichi; Makabe, Koki; Kuwajima, Kunihiro

2013-05-17

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.

Molecular Mechanisms of the Cytotoxicity of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET) and Other Protein-Oleic Acid Complexes*

PubMed Central

Nakamura, Takashi; Aizawa, Tomoyasu; Kariya, Ryusho; Okada, Seiji; Demura, Makoto; Kawano, Keiichi; Makabe, Koki; Kuwajima, Kunihiro

2013-01-01

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane. PMID:23580643

Versatility and Invariance in the Evolution of Homologous Heteromeric Interfaces

PubMed Central

Andreani, Jessica; Faure, Guilhem; Guerois, Raphaël

2012-01-01

Evolutionary pressures act on protein complex interfaces so that they preserve their complementarity. Nonetheless, the elementary interactions which compose the interface are highly versatile throughout evolution. Understanding and characterizing interface plasticity across evolution is a fundamental issue which could provide new insights into protein-protein interaction prediction. Using a database of 1,024 couples of close and remote heteromeric structural interologs, we studied protein-protein interactions from a structural and evolutionary point of view. We systematically and quantitatively analyzed the conservation of different types of interface contacts. Our study highlights astonishing plasticity regarding polar contacts at complex interfaces. It also reveals that up to a quarter of the residues switch out of the interface when comparing two homologous complexes. Despite such versatility, we identify two important interface descriptors which correlate with an increased conservation in the evolution of interfaces: apolar patches and contacts surrounding anchor residues. These observations hold true even when restricting the dataset to transiently formed complexes. We show that a combination of six features related either to sequence or to geometric properties of interfaces can be used to rank positions likely to share similar contacts between two interologs. Altogether, our analysis provides important tracks for extracting meaningful information from multiple sequence alignments of conserved binding partners and for discriminating near-native interfaces using evolutionary information. PMID:22952442

[The Role of Calcium in the Conformational Changes of the Recombinant S100A8/S100A9].

PubMed

Gheibi, N; Asghari, H; Chegini, K G; Sahmani, M; Moghadasi, M

2016-01-01

Calprotectin is a member of the EF-hand proteins, composed of two subunits, S100A8 (MRP8) and S100A9 (MRP14). These proteins are involved in important processes including cell signaling, regulation of inflammatory responses, cell cycle control, differentiation, regulation of ion channel activity and defense against microbial agents in a calcium dependent manner. In the present study, recombinant S100A8 and S100A9 were expressed in E. coli BL21 and then purified using Ni-NTA affinity chromatography. The structure of the S100A8/A9 complex in the presence and absence of calcium was assessed by circular dichroism and fluorescence spectroscopy. The intrinsic fluorescence emission spectra of the S100A8/A9 complex in the presence of calcium showed a reduction in fluorescence intensity, reflecting conformational changes within the protein with the exposure of aromatic residues to the protein surface. The far ultraviolet-circular dichroism spectra of the complex in the presence of calcium revealed minor changes in the regular secondary structure of the complex. Also, increased thermal stability of the S100A8/A9 complex in the presence of calcium was indicated.

Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx

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

Romano, Christine A.; Zhou, Mowei; Song, Yang

Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase, Mnx, in Bacillus sp. PL-12 is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. However, MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant tomore » crystallization, so its structure is unknown. In this study, native mass spectrometry defines the subunit topology and copper binding of the Mnx complex, while high resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for conceptualizing how Mnx produces nanoparticulate Mn oxides.« less

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.

PubMed Central

Liu, Y; Levit, M; Lurz, R; Surette, M G; Stock, J B

1997-01-01

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with approximately 14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor-kinase complexes within the cytoplasm. PMID:9405352

Not all transmembrane helices are born equal: Towards the extension of the sequence homology concept to membrane proteins

PubMed Central

2011-01-01

Background Sequence homology considerations widely used to transfer functional annotation to uncharacterized protein sequences require special precautions in the case of non-globular sequence segments including membrane-spanning stretches composed of non-polar residues. Simple, quantitative criteria are desirable for identifying transmembrane helices (TMs) that must be included into or should be excluded from start sequence segments in similarity searches aimed at finding distant homologues. Results We found that there are two types of TMs in membrane-associated proteins. On the one hand, there are so-called simple TMs with elevated hydrophobicity, low sequence complexity and extraordinary enrichment in long aliphatic residues. They merely serve as membrane-anchoring device. In contrast, so-called complex TMs have lower hydrophobicity, higher sequence complexity and some functional residues. These TMs have additional roles besides membrane anchoring such as intra-membrane complex formation, ligand binding or a catalytic role. Simple and complex TMs can occur both in single- and multi-membrane-spanning proteins essentially in any type of topology. Whereas simple TMs have the potential to confuse searches for sequence homologues and to generate unrelated hits with seemingly convincing statistical significance, complex TMs contain essential evolutionary information. Conclusion For extending the homology concept onto membrane proteins, we provide a necessary quantitative criterion to distinguish simple TMs (and a sufficient criterion for complex TMs) in query sequences prior to their usage in homology searches based on assessment of hydrophobicity and sequence complexity of the TM sequence segments. Reviewers This article was reviewed by Shamil Sunyaev, L. Aravind and Arcady Mushegian. PMID:22024092

Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways.

PubMed

Rouault, Tracey A; Maio, Nunziata

2017-08-04

Fe-S cofactors are composed of iron and inorganic sulfur in various stoichiometries. A complex assembly pathway conducts their initial synthesis and subsequent binding to recipient proteins. In this minireview, we discuss how discovery of the role of the mammalian cytosolic aconitase, known as iron regulatory protein 1 (IRP1), led to the characterization of the function of its Fe-S cluster in sensing and regulating cellular iron homeostasis. Moreover, we present an overview of recent studies that have provided insights into the mechanism of Fe-S cluster transfer to recipient Fe-S proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins.

PubMed

Rebière-Huët, J; Guérillon, J; Pimenta, A L; Di Martino, P; Orange, N; Hulen, C

2002-09-24

Bacterial adherence is a complex phenomenon involving specific interactions between receptors, including matricial fibronectin, and bacterial ligands. We show here that fibronectin and outer membrane proteins of Pseudomonas fluorescens were able to inhibit adherence of P. fluorescens to fibronectin-coated wells. We identified at least six fibronectin-binding proteins with molecular masses of 70, 55, 44, 37, 32 and 28 kDa. The presence of native (32 kDa) and heat-modified forms (37 kDa) of OprF was revealed by immuno-analysis and the 44-kDa band was composed of three proteins, their N-terminal sequences showing homologies with Pseudomonas aeruginosa porins (OprD, OprE1 and OprE3).

Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems*

PubMed Central

Durand, Eric; Zoued, Abdelrahim; Spinelli, Silvia; Watson, Paul J. H.; Aschtgen, Marie-Stéphanie; Journet, Laure; Cambillau, Christian; Cascales, Eric

2012-01-01

The Type VI secretion system (T6SS) is a macromolecular system distributed in Gram-negative bacteria, responsible for the secretion of effector proteins into target cells. The T6SS has a broad versatility as it can target both eukaryotic and prokaryotic cells. It is therefore involved in host pathogenesis or killing neighboring bacterial cells to colonize a new niche. At the architecture level, the T6SS core apparatus is composed of 13 proteins, which assemble in two subcomplexes. One of these subcomplexes, composed of subunits that share structural similarities with bacteriophage tail and baseplate components, is anchored to the cell envelope by the membrane subcomplex. This latter is constituted of at least three proteins, TssL, TssM, and TssJ. The crystal structure of the TssJ outer membrane lipoprotein and its interaction with the inner membrane TssM protein have been recently reported. TssL and TssM share sequence homology and characteristics with two components of the Type IVb secretion system (T4bSS), IcmH/DotU and IcmF, respectively. In this study, we report the crystal structure of the cytoplasmic domain of the TssL inner membrane protein from the enteroaggregative Escherichia coli Sci-1 T6SS. It folds as a hook-like structure composed of two three-helix bundles. Two TssL molecules associate to form a functional complex. Although the TssL trans-membrane segment is the main determinant of self-interaction, contacts between the cytoplasmic domains are required for TssL function. Based on sequence homology and secondary structure prediction, we propose that the TssL structure is the prototype for the members of the TssL and IcmH/DotU families. PMID:22371492

The neutralizing antibody response to the vaccinia virus A28 protein is specifically enhanced by its association with the H2 protein

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

Shinoda, Kaori; Wyatt, Linda S.; Moss, Bernard, E-mail: bmoss@niaid.nih.go

2010-09-15

The vaccinia virus (VACV) entry-fusion complex (EFC) is composed of at least nine membrane proteins. Immunization of mice with individual EFC genes induced corresponding protein-binding antibody but failed to protect against VACV intranasal challenge and only DNA encoding A28 elicited low neutralizing antibody. Because the A28 and H2 proteins interact, we determined the effect of immunizing with both genes simultaneously. This procedure greatly enhanced the amount of antibody that bound intact virions, neutralized infectivity, and provided partial protection against respiratory challenge. Neither injection of A28 and H2 plasmids at different sites or mixing A28 and H2 sera enhanced neutralizing antibody.more » The neutralizing antibody could be completely removed by binding to the A28 protein alone and the epitope was located in the C-terminal segment. These data suggest that the interaction of H2 with A28 stabilizes the immunogenic form of A28, mimicking an exposed region of the entry-fusion complex on infectious virions.« less

Combined Biology and Bioinformatics Approaches to Breast Cancer

DTIC Science & Technology

2005-04-01

interacted with the MH 1 and linker domains in both Smad3 and Smad4; no interaction was found with the MH2 domain (Fig. 7C). These data suggest that LMO4...LMO4 interacts with these Smads. GST protein-protein interaction assays showed that LMO4 binds to the MH1 and linker domain of Smad 1, Smad3 and Smad4...by facilitating the nuclear translocation and DNA-binding of a complex composed of an R-Smad (Smad2 and/or Smad3 ) and the co-Smad, Smad4 (10). To

Oligomers of Amyloid β Prevent Physiological Activation of the Cellular Prion Protein-Metabotropic Glutamate Receptor 5 Complex by Glutamate in Alzheimer Disease.

PubMed

Haas, Laura T; Strittmatter, Stephen M

2016-08-12

The dysfunction and loss of synapses in Alzheimer disease are central to dementia symptoms. We have recently demonstrated that pathological Amyloid β oligomer (Aβo) regulates the association between intracellular protein mediators and the synaptic receptor complex composed of cellular prion protein (PrP(C)) and metabotropic glutamate receptor 5 (mGluR5). Here we sought to determine whether Aβo alters the physiological signaling of the PrP(C)-mGluR5 complex upon glutamate activation. We provide evidence that acute exposure to Aβo as well as chronic expression of familial Alzheimer disease mutant transgenes in model mice prevents protein-protein interaction changes of the complex induced by the glutamate analog 3,5-dihydroxyphenylglycine. We further show that 3,5-dihydroxyphenylglycine triggers the phosphorylation and activation of protein-tyrosine kinase 2-β (PTK2B, also referred to as Pyk2) and of calcium/calmodulin-dependent protein kinase II in wild-type brain slices but not in Alzheimer disease transgenic brain slices or wild-type slices incubated with Aβo. This study further distinguishes two separate Aβo-dependent signaling cascades, one dependent on extracellular Ca(2+) and Fyn kinase activation and the other dependent on the release of Ca(2+) from intracellular stores. Thus, Aβo triggers multiple distinct PrP(C)-mGluR5-dependent events implicated in neurodegeneration and dementia. We propose that targeting the PrP(C)-mGluR5 complex will reverse aberrant Aβo-triggered states of the complex to allow physiological fluctuations of glutamate signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystal structure of the Csm3-Csm4 subcomplex in the type III-A CRISPR-Cas interference complex.

PubMed

Numata, Tomoyuki; Inanaga, Hideko; Sato, Chikara; Osawa, Takuo

2015-01-30

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci play a pivotal role in the prokaryotic host defense system against invading genetic materials. The CRISPR loci are transcribed to produce CRISPR RNAs (crRNAs), which form interference complexes with CRISPR-associated (Cas) proteins to target the invading nucleic acid for degradation. The interference complex of the type III-A CRISPR-Cas system is composed of five Cas proteins (Csm1-Csm5) and a crRNA, and targets invading DNA. Here, we show that the Csm1, Csm3, and Csm4 proteins from Methanocaldococcus jannaschii form a stable subcomplex. We also report the crystal structure of the M. jannaschii Csm3-Csm4 subcomplex at 3.1Å resolution. The complex structure revealed the presence of a basic concave surface around their interface, suggesting the RNA and/or DNA binding ability of the complex. A gel retardation analysis showed that the Csm3-Csm4 complex binds single-stranded RNA in a non-sequence-specific manner. Csm4 structurally resembles Cmr3, a component of the type III-B CRISPR-Cas interference complex. Based on bioinformatics, we constructed a model structure of the Csm1-Csm4-Csm3 ternary complex, which provides insights into its role in the Csm interference complex. Copyright © 2014 Elsevier Ltd. All rights reserved.

In vitro reconstitution and characterization of the yeast mitochondrial degradosome complex unravels tight functional interdependence.

PubMed

Malecki, Michal; Jedrzejczak, Robert; Stepien, Piotr P; Golik, Pawel

2007-09-07

The mitochondrial degradosome (mtEXO), the main RNA-degrading complex of yeast mitochondria, is composed of two subunits: an exoribonuclease encoded by the DSS1 gene and an RNA helicase encoded by the SUV3 gene. We expressed both subunits of the yeast mitochondrial degradosome in Escherichia coli, reconstituted the complex in vitro and analyzed the RNase, ATPase and helicase activities of the two subunits separately and in complex. The results reveal a very strong functional interdependence. For every enzymatic activity, we observed significant changes when the relevant protein was present in the complex, compared to the activity measured for the protein alone. The ATPase activity of Suv3p is stimulated by RNA and its background activity in the absence of RNA is reduced greatly when the protein is in the complex with Dss1p. The Suv3 protein alone does not display RNA-unwinding activity and the 3' to 5' directional helicase activity requiring a free 3' single-stranded substrate becomes apparent only when Suv3p is in complex with Dss1p. The Dss1 protein alone does have some basal exoribonuclease activity, which is not ATP-dependent, but in the presence of Suv3p the activity of the entire complex is enhanced greatly and is entirely ATP-dependent, with no residual activity observed in the absence of ATP. Such absolute ATP-dependence is unique among known exoribonuclease complexes. On the basis of these results, we propose a model in which the Suv3p RNA helicase acts as a molecular motor feeding the substrate to the catalytic centre of the RNase subunit.

Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes

PubMed Central

Sinars, Cindy R.; Cheung-Flynn, Joyce; Rimerman, Ronald A.; Scammell, Jonathan G.; Smith, David F.; Clardy, Jon

2003-01-01

The ability to bind immunosuppressive drugs such as cyclosporin and FK506 defines the immunophilin family of proteins, and the FK506-binding proteins form the FKBP subfamily of immunophilins. Some FKBPs, notably FKBP12 (the 12-kDa FK506-binding protein), have defined roles in regulating ion channels or cell signaling, and well established structures. Other FKBPs, especially the larger ones, participate in important biological processes, but their exact roles and the structural bases for these roles are poorly defined. FKBP51 (the 51-kDa FKBP) associates with heat shock protein 90 (Hsp90) and appears in functionally mature steroid receptor complexes. In New World monkeys, FKBP51 has been implicated in cortisol resistance. We report here the x-ray structures of human FKBP51, to 2.7 Å, and squirrel monkey FKBP51, to 2.8 Å, by using multiwavelength anomalous dispersion phasing. FKBP51 is composed of three domains: two consecutive FKBP domains and a three-unit repeat of the TPR (tetratricopeptide repeat) domain. This structure of a multi-FKBP domain protein clarifies the arrangement of these domains and their possible interactions with other proteins. The two FKBP domains differ by an insertion in the second that affects the formation of the progesterone receptor complex. PMID:12538866

ABRF-PRG07: advanced quantitative proteomics study.

PubMed

Falick, Arnold M; Lane, William S; Lilley, Kathryn S; MacCoss, Michael J; Phinney, Brett S; Sherman, Nicholas E; Weintraub, Susan T; Witkowska, H Ewa; Yates, Nathan A

2011-04-01

A major challenge for core facilities is determining quantitative protein differences across complex biological samples. Although there are numerous techniques in the literature for relative and absolute protein quantification, the majority is nonroutine and can be challenging to carry out effectively. There are few studies comparing these technologies in terms of their reproducibility, accuracy, and precision, and no studies to date deal with performance across multiple laboratories with varied levels of expertise. Here, we describe an Association of Biomolecular Resource Facilities (ABRF) Proteomics Research Group (PRG) study based on samples composed of a complex protein mixture into which 12 known proteins were added at varying but defined ratios. All of the proteins were present at the same concentration in each of three tubes that were provided. The primary goal of this study was to allow each laboratory to evaluate its capabilities and approaches with regard to: detection and identification of proteins spiked into samples that also contain complex mixtures of background proteins and determination of relative quantities of the spiked proteins. The results returned by 43 participants were compiled by the PRG, which also collected information about the strategies used to assess overall performance and as an aid to development of optimized protocols for the methodologies used. The most accurate results were generally reported by the most experienced laboratories. Among laboratories that used the same technique, values that were closer to the expected ratio were obtained by more experienced groups.

A novel method for purification of the endogenously expressed fission yeast Set2 complex.

PubMed

Suzuki, Shota; Nagao, Koji; Obuse, Chikashi; Murakami, Yota; Takahata, Shinya

2014-05-01

Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8μm), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation. Copyright © 2014 Elsevier Inc. All rights reserved.

Chaperonin polymers in archaea: The cytoskeleton of prokaryotes?

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

Trent, J.D.; Kagawa, H.K.; Zaluzec, N.J.

Chaperonins are protein complexes that play a critical role in folding nascent polypeptides under normal conditions and refolding damaged proteins under stress conditions. In all organisms these complexes are composed of evolutionarily conserved 60-kDa proteins arranged in double-ring structures with between 7 and 9 protein subunits per ring. These double ring structures are assumed to be the functional units in vivo, although they have never been observed inside cells. Here the authors show that the purified chaperonin from the hyperthermophilic archaeon Sulfolobus shibatae, which is closely related to chaperonins in eukaryotes, has a double ring structure at low concentrations (0.1more » mg/ml), but at more physiological concentrations, the rings stack end to end to form polymers. The polymers are stable at physiological temperatures (75 C) and closely resemble structures observed inside unfixed S. shibatae cells. The authors suggest that in vivo chaperonin activity may be regulated by polymerization and that chaperonin polymers may act as a cytoskeleton-like structure in archaea and bacteria.« less

Chaperonin Polymers in Archaea: The Cytoskeleton of Prokaryotes?

DOE R&D Accomplishments Database

Trent, J. D.; Kagawa, H. K.; Zaluzec, N. J.

1997-07-01

Chaperonins are protein complexes that play a critical role in folding nascent polypeptides under normal conditions and refolding damaged proteins under stress conditions. In all organisms these complexes are composed of evolutionarily conserved 60-kDa proteins arranged in double-ring structures with between 7 and 9 protein subunits per ring. These double ring structures are assumed to be the functional units in vivo, although they have never been observed inside cells. Here the authors show that the purified chaperonin from the hyperthermophilic archaeon Sulfolobus shibatae, which is closely related to chaperonins in eukaryotes, has a double ring structure at low concentrations (0.1 mg/ml), but at more physiological concentrations, the rings stack end to end to form polymers. The polymers are stable at physiological temperatures (75 C) and closely resemble structures observed inside unfixed S. shibatae cells. The authors suggest that in vivo chaperonin activity may be regulated by polymerization and that chaperonin polymers may act as a cytoskeleton-like structure in archaea and bacteria.

Structure of the Putative 32 kDa Myrosinase Binding Protein from Arabidopsis (At3g16450.1) Determined by SAIL-NMR

PubMed Central

Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira Mei; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, JunGoo; Güntert, Peter; Aceti, David J.; Markley, John L.; Kainosho, Masatsune

2009-01-01

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme, myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniformly 13C/15N labeling methods, we used our stereo-array isotope labeling (SAIL) technology to prepare an optimally 2H/13C/15N-labeled sample. NMR data sets collected with the SAIL-protein enabled us to assign 1H, 13C and 15N chemical shifts to 95.5% of all atoms, even at the low concentration (0.2 mM) of the protein product. We collected additional NOESY data and solved the three-dimensional structure with the CYANA software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent, but similar, lectin-fold domains composed of three β-sheets. PMID:19021763

Structure of the putative 32 kDa myrosinase-binding protein from Arabidopsis (At3g16450.1) determined by SAIL-NMR.

PubMed

Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira M; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, Jungoo; Güntert, Peter; Aceti, David J; Markley, John L; Kainosho, Masatsune

2008-12-01

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform (13)C/(15)N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally (2)H/(13)C/(15)N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign (1)H, (13)C and (15)N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three beta-sheets.

Tricomponent Immunopotentiating System as a Novel Molecular Design Strategy for Malaria Vaccine Development ▿

PubMed Central

Miyata, Takeshi; Harakuni, Tetsuya; Tsuboi, Takafumi; Sattabongkot, Jetsumon; Ikehara, Ayumu; Tachibana, Mayumi; Torii, Motomi; Matsuzaki, Goro; Arakawa, Takeshi

2011-01-01

The creation of subunit vaccines to prevent malaria infection has been hampered by the intrinsically weak immunogenicity of the recombinant antigens. We have developed a novel strategy to increase immune responses by creating genetic fusion proteins to target specific antigen-presenting cells (APCs). The fusion complex was composed of three physically linked molecular entities: (i) a vaccine antigen, (ii) a multimeric α-helical coiled-coil core, and (iii) an APC-targeting ligand linked to the core via a flexible linker. The vaccine efficacy of the tricomponent complex was evaluated using an ookinete surface protein of Plasmodium vivax, Pvs25, and merozoite surface protein-1 of Plasmodium yoelii. Immunization of mice with the tricomponent complex induced a robust antibody response and conferred substantial levels of P. vivax transmission blockade as evaluated by a membrane feed assay, as well as protection from lethal P. yoelii infection. The observed effect was strongly dependent on the presence of all three components physically integrated as a fusion complex. This system, designated the tricomponent immunopotentiating system (TIPS), onto which any recombinant protein antigens or nonproteinaceous substances could be loaded, may be a promising strategy for devising subunit vaccines or adjuvants against various infectious diseases, including malaria. PMID:21807905

A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion

DOE PAGES

Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko

2014-11-06

Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components thatmore » form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.« less

Microscopic insight into thermodynamics of conformational changes of SAP-SLAM complex in signal transduction cascade

NASA Astrophysics Data System (ADS)

Samanta, Sudipta; Mukherjee, Sanchita

2017-04-01

The signalling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, associate with SLAM-associated protein (SAP)-related molecules, composed of single SH2 domain architecture. SAP activates Src-family kinase Fyn after SLAM ligation, resulting in a SLAM-SAP-Fyn complex, where, SAP binds the Fyn SH3 domain that does not involve canonical SH3 or SH2 interactions. This demands insight into this SAP mediated signalling cascade. Thermodynamics of the conformational changes are extracted from the histograms of dihedral angles obtained from the all-atom molecular dynamics simulations of this structurally well characterized SAP-SLAM complex. The results incorporate the binding induced thermodynamic changes of individual amino acid as well as the secondary structural elements of the protein and the solvent. Stabilization of the peptide partially comes through a strong hydrogen bonding network with the protein, while hydrophobic interactions also play a significant role where the peptide inserts itself into a hydrophobic cavity of the protein. SLAM binding widens SAP's second binding site for Fyn, which is the next step in the signal transduction cascade. The higher stabilization and less fluctuation of specific residues of SAP in the Fyn binding site, induced by SAP-SLAM complexation, emerge as the key structural elements to trigger the recognition of SAP by the SH3 domain of Fyn. The thermodynamic quantification of the protein due to complexation not only throws deeper understanding in the established mode of SAP-SLAM interaction but also assists in the recognition of the relevant residues of the protein responsible for alterations in its activity.

Microscopic insight into thermodynamics of conformational changes of SAP-SLAM complex in signal transduction cascade.

PubMed

Samanta, Sudipta; Mukherjee, Sanchita

2017-04-28

The signalling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, associate with SLAM-associated protein (SAP)-related molecules, composed of single SH2 domain architecture. SAP activates Src-family kinase Fyn after SLAM ligation, resulting in a SLAM-SAP-Fyn complex, where, SAP binds the Fyn SH3 domain that does not involve canonical SH3 or SH2 interactions. This demands insight into this SAP mediated signalling cascade. Thermodynamics of the conformational changes are extracted from the histograms of dihedral angles obtained from the all-atom molecular dynamics simulations of this structurally well characterized SAP-SLAM complex. The results incorporate the binding induced thermodynamic changes of individual amino acid as well as the secondary structural elements of the protein and the solvent. Stabilization of the peptide partially comes through a strong hydrogen bonding network with the protein, while hydrophobic interactions also play a significant role where the peptide inserts itself into a hydrophobic cavity of the protein. SLAM binding widens SAP's second binding site for Fyn, which is the next step in the signal transduction cascade. The higher stabilization and less fluctuation of specific residues of SAP in the Fyn binding site, induced by SAP-SLAM complexation, emerge as the key structural elements to trigger the recognition of SAP by the SH3 domain of Fyn. The thermodynamic quantification of the protein due to complexation not only throws deeper understanding in the established mode of SAP-SLAM interaction but also assists in the recognition of the relevant residues of the protein responsible for alterations in its activity.

Self-assembling enzymes and the origins of the cytoskeleton

PubMed Central

Barry, Rachael; Gitai, Zemer

2011-01-01

The bacterial cytoskeleton is composed of a complex and diverse group of proteins that self-assemble into linear filaments. These filaments support and organize cellular architecture and provide a dynamic network controlling transport and localization within the cell. Here, we review recent discoveries related to a newly appreciated class of self-assembling proteins that expand our view of the bacterial cytoskeleton and provide potential explanations for its evolutionary origins. Specifically, several types of metabolic enzymes can form structures similar to established cytoskeletal filaments and, in some cases, these structures have been repurposed for structural uses independent of their normal role. The behaviors of these enzymes suggest that some modern cytoskeletal proteins may have evolved from dual-role proteins with catalytic and structural functions. PMID:22014508

Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

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

Raymond, Amy; Lovell, Scott; Lorimer, Don

2009-12-01

With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. colimore » and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.« less

Fluorescence study of protein-lipid complexes with a new symmetric squarylium probe.

PubMed

Ioffe, Valeriya M; Gorbenko, Galyna P; Deligeorgiev, Todor; Gadjev, Nikolai; Vasilev, Aleksey

2007-06-01

The novel symmetric squarylium derivative SQ-1 has been synthesized and tested for its sensitivity to the formation of protein-lipid complexes. SQ-1 binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) exerted an influence on the probe association with lipid vesicles resulting presumably from the competition between SQ-1 and the proteins for bilayer free volume and modification of its properties. The magnitude of this effect was much higher for lysozyme which may stem from the amphipathy of protein alpha-helix involved in the membrane binding. Varying membrane composition provides evidence for the dye sensitivity to both hydrophobic and electrostatic protein-lipid interactions. Fluorescence anisotropy studies uncovered the restriction of SQ-1 rotational mobility in lipid environment in the presence of Lz and RNase being indicative of the incorporation of the proteins into bilayer interior. The results of binding, fluorescence quenching and kinetic experiments suggested lysozyme-induced local lipid demixing upon protein association with negatively charged membranes with threshold concentration of CL for the lipid demixing being 10 mol%.

Modeling of protein binary complexes using structural mass spectrometry data

PubMed Central

Kamal, J.K. Amisha; Chance, Mark R.

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. PMID:18042684

Structural complexity and molecular heterogeneity of a butterfly ejaculate reflect a complex history of selection

PubMed Central

Cherwin, Tamara S.; Plakke, Melissa S.; Hill, Jason; Small, Brandon S.; Goetz, Breanna J.; Wheat, Christopher W.; Morehouse, Nathan I.

2017-01-01

Male ejaculates are often structurally complex, and this complexity is likely to influence key reproductive interactions between males and females. However, despite its potential evolutionary significance, the molecular underpinnings of ejaculate structural complexity have received little empirical attention. To address this knowledge gap, we sought to understand the biochemical and functional properties of the structurally complex ejaculates of Pieris rapae butterflies. Males in this species produce large ejaculates called spermatophores composed of an outer envelope, an inner matrix, and a bolus of sperm. Females are thought to benefit from the nutrition contained in the soluble inner matrix through increases in longevity and fecundity. However, the indigestible outer envelope of the spermatophore delays female remating, allowing males to monopolize paternity for longer. Here, we show that these two nonsperm-containing spermatophore regions, the inner matrix and the outer envelope, differ in their protein composition and functional properties. We also reveal how these divergent protein mixtures are separately stored in the male reproductive tract and sequentially transferred to the female reproductive tract during spermatophore assembly. Intriguingly, we discovered large quantities of female-derived proteases in both spermatophore regions shortly after mating, which may contribute to spermatophore digestion and hence, female control over remating rate. Finally, we report evidence of past selection on these spermatophore proteins and female proteases, indicating a complex evolutionary history. Our findings illustrate how structural complexity of ejaculates may allow functionally and/or spatially associated suites of proteins to respond rapidly to divergent selective pressures, such as sexual conflict or reproductive cooperation. PMID:28630352

The Mini-Chromosome Maintenance (Mcm) Complexes Interact with DNA Polymerase α-Primase and Stimulate Its Ability to Synthesize RNA Primers

PubMed Central

You, Zhiying; De Falco, Mariarosaria; Kamada, Katsuhiko; Pisani, Francesca M.; Masai, Hisao

2013-01-01

The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes. PMID:23977294

Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family

PubMed Central

Uhart, Marina; Flores, Gabriel; Bustos, Diego M.

2016-01-01

Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

Matricellular proteins in drug delivery: Therapeutic targets, active agents, and therapeutic localization.

PubMed

Sawyer, Andrew J; Kyriakides, Themis R

2016-02-01

Extracellular matrix is composed of a complex array of molecules that together provide structural and functional support to cells. These properties are mainly mediated by the activity of collagenous and elastic fibers, proteoglycans, and proteins such as fibronectin and laminin. ECM composition is tissue-specific and could include matricellular proteins whose primary role is to modulate cell-matrix interactions. In adults, matricellular proteins are primarily expressed during injury, inflammation and disease. Particularly, they are closely associated with the progression and prognosis of cardiovascular and fibrotic diseases, and cancer. This review aims to provide an overview of the potential use of matricellular proteins in drug delivery including the generation of therapeutic agents based on the properties and structures of these proteins as well as their utility as biomarkers for specific diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.

PubMed

Castelle, Cindy J; Roger, Magali; Bauzan, Marielle; Brugna, Myriam; Lignon, Sabrina; Nimtz, Manfred; Golyshina, Olga V; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne

2015-08-01

The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel Insights into the Role of Neurospora crassa NDUFAF2, an Evolutionarily Conserved Mitochondrial Complex I Assembly Factor

PubMed Central

Pereira, Bruno; Videira, Arnaldo

2013-01-01

Complex I deficiency is commonly associated with mitochondrial oxidative phosphorylation diseases. Mutations in nuclear genes encoding structural subunits or assembly factors of complex I have been increasingly identified as the cause of the diseases. One such factor, NDUFAF2, is a paralog of the NDUFA12 structural subunit of the enzyme, but the mechanism by which it exerts its function remains unknown. Herein, we demonstrate that the Neurospora crassa NDUFAF2 homologue, the 13.4L protein, is a late assembly factor that associates with complex I assembly intermediates containing the membrane arm and the connecting part but lacking the N module of the enzyme. Furthermore, we provide evidence that dissociation of the assembly factor is dependent on the incorporation of the putative regulatory module composed of the subunits of 13.4 (NDUFA12), 18.4 (NDUFS6), and 21 (NDUFS4) kDa. Our results demonstrate that the 13.4L protein is a complex I assembly factor functionally conserved from fungi to mammals. PMID:23648483

The costa of trichomonads: A complex macromolecular cytoskeleton structure made of uncommon proteins.

PubMed

de Andrade Rosa, Ivone; Caruso, Marjolly Brigido; de Oliveira Santos, Eidy; Gonzaga, Luiz; Zingali, Russolina Benedeta; de Vasconcelos, Ana Tereza R; de Souza, Wanderley; Benchimol, Marlene

2017-06-01

The costa is a prominent striated fibre that is found in protozoa of the Trichomonadidae family that present an undulating membrane. It is composed primarily of proteins that have not yet been explored. In this study, we used cell fractionation to obtain a highly enriched costa fraction whose structure and composition was further analysed by electron microscopy and mass spectrometry. Electron microscopy of negatively stained samples revealed that the costa, which is a periodic structure with alternating electron-dense and electron-lucent bands, displays three distinct regions, named the head, neck and body. Fourier transform analysis showed that the electron-lucent bands present sub-bands with a regular pattern. An analysis of the costa fraction via one- and two-dimensional electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) allowed the identification of 54 hypothetical proteins. Fourteen of those proteins were considered to be major components of the fraction. The costa of T. foetus is a complex and organised cytoskeleton structure made of a large number of proteins which is assembled into filamentous structures. Some of these proteins exhibit uncharacterised domains and no function related according to gene ontology, suggesting that the costa structure may be formed by a new class of proteins that differ from those previously described in other organisms. Seven of these proteins contain prefoldin domains displaying coiled-coil regions. This propriety is shared with proteins of the striated fibres of other protozoan as well as in intermediate filaments. Our observations suggest the presence of a new class of the cytoskeleton filaments in T. foetus. We believe that our data could auxiliate in determining the specific locations of these proteins in the distinct regions that compose the costa, as well as to define the functional roles of each component. Therefore, our study will help in the better understanding of the organisation and function of this structure in unicellular organisms. © 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface.

PubMed

Fleming, Patrick J; Patel, Dhilon S; Wu, Emilia L; Qi, Yifei; Yeom, Min Sun; Sousa, Marcelo Carlos; Fleming, Karen G; Im, Wonpil

2016-06-21

The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Isolation of monomeric photosystem II that retains the subunit PsbS.

PubMed

Haniewicz, Patrycja; De Sanctis, Daniele; Büchel, Claudia; Schröder, Wolfgang P; Loi, Maria Cecilia; Kieselbach, Thomas; Bochtler, Matthias; Piano, Dario

2013-12-01

Photosystem II has been purified from a transplastomic strain of Nicotiana tabacum according to two different protocols. Using the procedure described in Piano et al. (Photosynth Res 106:221-226, 2010) it was possible to isolate highly active PSII composed of monomers and dimers but depleted in their PsbS protein content. A "milder" procedure than the protocol reported by Fey et al. (Biochim Biophys Acta 1777:1501-1509, 2008) led to almost exclusively monomeric PSII complexes which in part still bind the PsbS protein. This finding might support a role for PSII monomers in higher plants.

CMC determination of nonionic surfactants in protein formulations using ultrasonic resonance technology.

PubMed

Horiuchi, Shohei; Winter, Gerhard

2015-05-01

Biological products often contain surfactants as stabilizers in their formulations to avoid surface adsorption, interfacial denaturation and aggregation of the protein drug and thereby improve the overall pharmaceutical quality of the product. On the other hand, when the surfactant concentration exceeds the critical micelle concentration (CMC) in a protein formulation, protein-loaded micelles could be formed which could potentially be the cause of immunogenicity. Therefore, the actual CMC and the presence of micelles generally need to be confirmed for each protein formulation because the CMC is affected by the presence of protein and other formulation factors. In this study, the ultrasonic resonance technology (URT) was applied to determine CMC of surfactants in pharmaceutical protein solutions in comparison with surface tensiometry (TE) and dynamic light scattering (DLS). According to our results, the ultrasonic resonance technology can easily and precisely provide CMCs of surfactants in protein formulations while it is not working for protein-free formulations. This indicates that the signal we measure with ultrasonic velocity comes from complex micelles composed of surfactant and protein molecules. DLS did not provide reliable data for protein/surfactant systems. Interestingly, a protein formulation with arginine and polysorbate 20 behaved differently when studied with TE and URT allowing us to see that arginine is bound to protein and that the complex interacts with the surfactant. Copyright © 2015 Elsevier B.V. All rights reserved.

Unfolding and refolding details of lysozyme in the presence of β-casein micelles.

PubMed

Wu, Fu-Gen; Luo, Jun-Jie; Yu, Zhi-Wu

2011-02-28

In this work, we selected a small globular protein, lysozyme, to study how it unfolds and refolds in the presence of micelles composed of the unstructured β-casein proteins by using microcalorimetry and circular dichroism spectroscopy. It was found that a partially unfolded structure of lysozyme starts to form when the β-casein/lysozyme molar ratio is above 0.7, and the structure forms exclusively when the β-casein/lysozyme molar ratio is above 1.6. This partially unfolded state of lysozyme loses most of its tertiary structure and after heating, the denatured lysozyme molecules are trapped in the charged coatings of β-casein micelles and cannot refold upon cooling. The thus obtained protein complex can be viewed as a kind of special polyelectrolyte complex micelle. The net charge ratios of the two proteins and the ionic strength of the dispersions can significantly modulate the electrostatic and hydrophobic interactions between the two proteins. Our present work may have implications for the nanoparticle protein engineering therapy in the biomedicine field and may provide a better understanding of the principles governing the protein-protein interactions. Besides, the heating-cooling-reheating procedure employed in this work can also be used to study the unfolding and refolding details of the target protein in other protein-protein, protein-polymer and protein-small solute systems.

Bipartite design of a self-fibrillating protein copolymer with nanopatterned peptide display capabilities.

PubMed

Bruning, Marc; Kreplak, Laurent; Leopoldseder, Sonja; Müller, Shirley A; Ringler, Philippe; Duchesne, Laurence; Fernig, David G; Engel, Andreas; Ucurum-Fotiadis, Zöhre; Mayans, Olga

2010-11-10

The development of biomatrices for technological and biomedical applications employs self-assembled scaffolds built from short peptidic motifs. However, biopolymers composed of protein domains would offer more varied molecular frames to introduce finer and more complex functionalities in bioreactive scaffolds using bottom-up approaches. Yet, the rules governing the three-dimensional organization of protein architectures in nature are complex and poorly understood. As a result, the synthetic fabrication of ordered protein association into polymers poses major challenges to bioengineering. We have now fabricated a self-assembling protein nanofiber with predictable morphologies and amenable to bottom-up customization, where features supporting function and assembly are spatially segregated. The design was inspired by the cross-linking of titin filaments by telethonin in the muscle sarcomere. The resulting fiber is a two-protein system that has nanopatterned peptide display capabilities as shown by the recruitment of functionalized gold nanoparticles at regular intervals of ∼ 5 nm, yielding a semiregular linear array over micrometers. This polymer promises the uncomplicated display of biologically active motifs to selectively bind and organize matter in the fine nanoscale. Further, its conceptual design has high potential for controlled plurifunctionalization.

The ribonucleoprotein Csr network.

PubMed

Seyll, Ethel; Van Melderen, Laurence

2013-11-08

Ribonucleoprotein complexes are essential regulatory components in bacteria. In this review, we focus on the carbon storage regulator (Csr) network, which is well conserved in the bacterial world. This regulatory network is composed of the CsrA master regulator, its targets and regulators. CsrA binds to mRNA targets and regulates translation either negatively or positively. Binding to small non-coding RNAs controls activity of this protein. Expression of these regulators is tightly regulated at the level of transcription and stability by various global regulators (RNAses, two-component systems, alarmone). We discuss the implications of these complex regulations in bacterial adaptation.

LILBID-mass spectrometry of the mitochondrial preprotein translocase TOM.

PubMed

Mager, Frauke; Sokolova, Lucie; Lintzel, Julia; Brutschy, Bernhard; Nussberger, Stephan

2010-11-17

In the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) to the outer mitochondrial membrane protein translocon TOM to analyze its subunit composition and stoichiometry. With TOM core complex, purified at high pH, we demonstrate that a TOM core complex of Neurospora crassa is composed of at least two Tom40 and Tom22 molecules, respectively, and more than five small Tom subunits between 5.5 and 6.4 kDa. We show that the multiprotein complex has a total molecular mass higher than 170 depending on the number of Tom5, Tom6 and Tom7 molecules bound.

LILBID-mass spectrometry of the mitochondrial preprotein translocase TOM

NASA Astrophysics Data System (ADS)

Mager, Frauke; Sokolova, Lucie; Lintzel, Julia; Brutschy, Bernhard; Nussberger, Stephan

2010-11-01

In the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) to the outer mitochondrial membrane protein translocon TOM to analyze its subunit composition and stoichiometry. With TOM core complex, purified at high pH, we demonstrate that a TOM core complex of Neurospora crassa is composed of at least two Tom40 and Tom22 molecules, respectively, and more than five small Tom subunits between 5.5 and 6.4 kDa. We show that the multiprotein complex has a total molecular mass higher than 170 depending on the number of Tom5, Tom6 and Tom7 molecules bound.

Lysosomal multienzyme complex: pros and cons of working together.

PubMed

Bonten, Erik J; Annunziata, Ida; d'Azzo, Alessandra

2014-06-01

The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, protective protein/cathepsin A (PPCA), the sialidase, neuraminidase-1 (NEU1), and the glycosidase β-galactosidase (β-GAL). Next to this 'core' complex, the existence of sub-complexes, which may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1, and β-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders.

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

PubMed

Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

2012-05-01

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe–S Assembly Complex

PubMed Central

Fox, Nicholas G.; Das, Deepika; Chakrabarti, Mrinmoy; Lindahl, Paul A.; Barondeau, David P.

2015-01-01

Iron–sulfur (Fe–S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe–S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe–S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe–S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe–S assembly complex. Here the kinetics of Fe–S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe–S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe–S assembly complex. PMID:26016518

Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe-S Assembly Complex.

PubMed

Fox, Nicholas G; Das, Deepika; Chakrabarti, Mrinmoy; Lindahl, Paul A; Barondeau, David P

2015-06-30

Iron-sulfur (Fe-S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe-S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe-S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe-S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe-S assembly complex. Here the kinetics of Fe-S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe-S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe-S assembly complex.

Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology.

PubMed

Palomares, Laura A; Srivastava, Indresh K; Ramírez, Octavio T; Cox, Manon M J

2018-06-10

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.

The cellulose-binding activity of the PsB multiprotein complex is required for proper assembly of the spore coat and spore viability in Dictyostelium discoideum.

PubMed

Srinivasan, S; Griffiths, K R; McGuire, V; Champion, A; Williams, K L; Alexander, S

2000-08-01

The terminal event of spore differentiation in the cellular slime mould Dictyostelium discoideum is the assembly of the spore coat, which surrounds the dormant amoeba and allows the organism to survive during extended periods of environmental stress. The spore coat is a polarized extracellular matrix composed of glycoproteins and cellulose. The process of spore coat formation begins by the regulated secretion of spore coat proteins from the prespore vesicles (PSVs). Four of the major spore coat proteins (SP96, PsB/SP85, SP70 and SP60) exist as a preassembled multiprotein complex within the PSVs. This complete complex has an endogenous cellulose-binding activity. Mutant strains lacking either the SP96 or SP70 proteins produce partial complexes that do not have cellulose-binding activity, while mutants lacking SP60 produce a partial complex that retains this activity. Using a combination of immunofluorescence microscopy and biochemical methods we now show that the lack of cellulose-binding activity in the SP96 and SP70 mutants results in abnormally assembled spore coats and spores with greatly reduced viability. In contrast, the SP60 mutant, in which the PsB complex retains its cellulose-binding activity, produces spores with apparently unaltered structure and viability. Thus, it is the loss of the cellulose-binding activity of the PsB complex, rather than the mere loss of individual spore coat proteins, that results in compromised spore coat structure. These results support the idea that the cellulose-binding activity associated with the complete PsB complex plays an active role in the assembly of the spore coat.

CRISPR RNA and anti-CRISPR protein binding to the Xanthomonas albilineans Csy1-Csy2 heterodimer in the type I-F CRISPR-Cas system.

PubMed

Hong, Suji; Ka, Donghyun; Yoon, Seo Jeong; Suh, Nayoung; Jeong, Migyeong; Suh, Jeong-Yong; Bae, Euiyoung

2018-02-23

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide microbial adaptive immunity against bacteriophages. In type I-F CRISPR-Cas systems, multiple Cas proteins (Csy1-4) compose a surveillance complex (Csy complex) with CRISPR RNA (crRNA) for target recognition. Here, we report the biochemical characterization of the Csy1-Csy2 subcomplex from Xanthomonas albilineans , including the analysis of its interaction with crRNA and AcrF2, an anti-CRISPR (Acr) protein from a phage that infects Pseudomonas aeruginosa The X. albilineans Csy1 and Csy2 proteins (XaCsy1 and XaCsy2, respectively) formed a stable heterodimeric complex that specifically bound the 8-nucleotide (nt) 5'-handle of the crRNA. In contrast, the XaCsy1-XaCsy2 heterodimer exhibited reduced affinity for the 28-nt X. albilineans CRISPR repeat RNA containing the 5'-handle sequence. Chromatographic and calorimetric analyses revealed tight binding between the Acr protein from the P. aeruginosa phage and the heterodimeric subunit of the X. albilineans Csy complex, suggesting that AcrF2 recognizes conserved features of Csy1-Csy2 heterodimers. We found that neither XaCsy1 nor XaCsy2 alone forms a stable complex with AcrF2 and the 5'-handle RNA, indicating that XaCsy1-XaCsy2 heterodimerization is required for binding them. We also solved the crystal structure of AcrF2 to a resolution of 1.34 Å, enabling a more detailed structural analysis of the residues involved in the interactions with the Csy1-Csy2 heterodimer. Our results provide information about the order of events during the formation of the multisubunit crRNA-guided surveillance complex and suggest that the Acr protein inactivating type I-F CRISPR-Cas systems has broad specificity. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Two-dimensional turbulent flow chromatography coupled on-line to liquid chromatography-mass spectrometry for solution-based ligand screening against multiple proteins.

PubMed

Zhou, Jian-Liang; An, Jing-Jing; Li, Ping; Li, Hui-Jun; Jiang, Yan; Cheng, Jie-Fei

2009-03-20

We present herein a novel bioseparation/chemical analysis strategy for protein-ligand screening and affinity ranking in compound mixtures, designed to increase screening rates and improve sensitivity and ruggedness in performance. The strategy is carried out by combining on-line two-dimensional turbulent flow chromatography (2D-TFC) with liquid chromatography-mass spectrometry (LC-MS), and accomplished through the following steps: (1) a reversed-phase TFC stage to separate the protein/ligand complex from the unbound free molecules, (2) an on-line dissociation process to release the bound ligands from the complexes, and (3) a second mixed-mode cation-exchange/reversed-phase TFC stage to trap the bound ligands and to remove the proteins and salts, followed by LC-MS analysis for identification and determination of the binding affinities. The technique can implement an ultra-fast isolation of protein/ligand complex with the retention time of a complex peak in about 5s, and on-line prepare the "clean" sample to be directly compatible with the LC-MS analysis. The improvement in performance of this 2D-TFC/LC-MS approach over the conventional approach has been demonstrated by determining affinity-selected ligands of the target proteins acetylcholinesterase and butyrylcholinesterase from a small library with known binding affinities and a steroidal alkaloid library composed of structurally similar compounds. Our results show that 2D-TFC/LC-MS is a generic and efficient tool for high-throughput screening of ligands with low-to-high binding affinities, and structure-activity relationship evaluation.

Structural characterization of the Yersinia pestis type III secretion system needle protein YscF in complex with its heterodimeric chaperone YscE/YscG

PubMed Central

Sun, Ping; Tropea, Joseph E.; Austin, Brian P.; Cherry, Scott; Waugh, David S.

2008-01-01

Summary The plague-causing bacterium Yersinia pestis utilizes a Type III Secretion System (T3SS) to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as "chaperones" to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 Å resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa T3SS, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat (TPR) family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal TPR motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the N-terminal 49 residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex. PMID:18281060

Structural Characterization of the Yersinia pestis Type III Secretion System Needle Protein YscF in Complex with Its Heterodimeric Chaperone YscE/YscG

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

Sun, Ping; Tropea, Joseph E.; Austin, Brian P.

2008-05-03

The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as 'chaperones' to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 {angstrom} resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonasmore » aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.« less

GHSR-D2R heteromerization modulates dopamine signaling through an effect on G protein conformation.

PubMed

Damian, Marjorie; Pons, Véronique; Renault, Pedro; M'Kadmi, Céline; Delort, Bartholomé; Hartmann, Lucie; Kaya, Ali I; Louet, Maxime; Gagne, Didier; Ben Haj Salah, Khoubaib; Denoyelle, Séverine; Ferry, Gilles; Boutin, Jean A; Wagner, Renaud; Fehrentz, Jean-Alain; Martinez, Jean; Marie, Jacky; Floquet, Nicolas; Galès, Céline; Mary, Sophie; Hamm, Heidi E; Banères, Jean-Louis

2018-04-24

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.

Structure of a Type-1 Secretion System ABC Transporter.

PubMed

Morgan, Jacob L W; Acheson, Justin F; Zimmer, Jochen

2017-03-07

Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-Å crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access. Copyright © 2017 Elsevier Ltd. All rights reserved.

LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit.

PubMed

Fowler, Sandy; Maguin, Pascal; Kalan, Sampada; Loayza, Diego

2018-06-22

DNA damage response pathways are essential for genome stability and cell survival. Specifically, the ATR kinase is activated by DNA replication stress. An early event in this activation is the recruitment and phosphorylation of RPA, a single stranded DNA binding complex composed of three subunits, RPA70, RPA32 and RPA14. We have previously shown that the LIM protein Ajuba associates with RPA, and that depletion of Ajuba leads to potent activation of ATR. In this study, we provide evidence that the Ajuba-RPA interaction occurs through direct protein contact with RPA70, and that their association is cell cycle-regulated and is reduced upon DNA replication stress. We propose a model in which Ajuba negatively regulates the ATR pathway by directly interacting with RPA70, thereby preventing inappropriate ATR activation. Our results provide a framework to further our understanding of the mechanism of ATR regulation in human cells in the context of cellular transformation.

Dimerization of the Bacterial Biotin Carboxylase Subunit Is Required for Acetyl Coenzyme A Carboxylase Activity In Vivo

PubMed Central

Smith, Alexander C.

2012-01-01

Acetyl coenzyme A (acteyl-CoA) carboxylase (ACC) is the first committed enzyme of the fatty acid synthesis pathway. Escherichia coli ACC is composed of four different proteins. The first enzymatic activity of the ACC complex, biotin carboxylase (BC), catalyzes the carboxylation of the protein-bound biotin moiety of another subunit with bicarbonate in an ATP-dependent reaction. Although BC is found as a dimer in cell extracts and the carboxylase activities of the two subunits of the dimer are interdependent, mutant BC proteins deficient in dimerization are reported to retain appreciable activity in vitro (Y. Shen, C. Y. Chou, G. G. Chang, and L. Tong, Mol. Cell 22:807–818, 2006). However, in vivo BC must interact with the other proteins of the complex, and thus studies of the isolated BC may not reflect the intracellular function of the enzyme. We have tested the abilities of three BC mutant proteins deficient in dimerization to support growth and report that the two BC proteins most deficient in dimerization fail to support growth unless expressed at high levels. In contrast, the wild-type protein supports growth at low expression levels. We conclude that BC must be dimeric to fulfill its physiological function. PMID:22037404

Deconstructing the core dynamics from a complex time-lagged regulatory biological circuit.

PubMed

Eriksson, O; Brinne, B; Zhou, Y; Björkegren, J; Tegnér, J

2009-03-01

Complex regulatory dynamics is ubiquitous in molecular networks composed of genes and proteins. Recent progress in computational biology and its application to molecular data generate a growing number of complex networks. Yet, it has been difficult to understand the governing principles of these networks beyond graphical analysis or extensive numerical simulations. Here the authors exploit several simplifying biological circumstances which thereby enable to directly detect the underlying dynamical regularities driving periodic oscillations in a dynamical nonlinear computational model of a protein-protein network. System analysis is performed using the cell cycle, a mathematically well-described complex regulatory circuit driven by external signals. By introducing an explicit time delay and using a 'tearing-and-zooming' approach the authors reduce the system to a piecewise linear system with two variables that capture the dynamics of this complex network. A key step in the analysis is the identification of functional subsystems by identifying the relations between state-variables within the model. These functional subsystems are referred to as dynamical modules operating as sensitive switches in the original complex model. By using reduced mathematical representations of the subsystems the authors derive explicit conditions on how the cell cycle dynamics depends on system parameters, and can, for the first time, analyse and prove global conditions for system stability. The approach which includes utilising biological simplifying conditions, identification of dynamical modules and mathematical reduction of the model complexity may be applicable to other well-characterised biological regulatory circuits. [Includes supplementary material].

Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex.

PubMed

Cardote, Teresa A F; Gadd, Morgan S; Ciulli, Alessio

2017-06-06

Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2 VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Real-Time Kinetic Probes Support Monothiol Glutaredoxins As Intermediate Carriers in Fe-S Cluster Biosynthetic Pathways.

PubMed

Vranish, James N; Das, Deepika; Barondeau, David P

2016-11-18

Iron-sulfur (Fe-S) clusters are protein cofactors that are required for many essential cellular functions. Fe-S clusters are synthesized and inserted into target proteins by an elaborate biosynthetic process. The insensitivity of most Fe-S assembly and transfer assays requires high concentrations for components and places major limits on reaction complexity. Recently, fluorophore labels were shown to be effective at reporting cluster content for Fe-S proteins. Here, the incorporation of this labeling approach allowed the design and interrogation of complex Fe-S cluster biosynthetic reactions that mimic in vivo conditions. A bacterial Fe-S assembly complex, composed of the cysteine desulfurase IscS and scaffold protein IscU, was used to generate [2Fe-2S] clusters for transfer to mixtures of putative intermediate carrier and acceptor proteins. The focus of this study was to test whether the monothiol glutaredoxin, Grx4, functions as an obligate [2Fe-2S] carrier protein in the Fe-S cluster distribution network. Interestingly, [2Fe-2S] clusters generated by the IscS-IscU complex transferred to Grx4 at rates comparable to previous assays using uncomplexed IscU as a cluster source in chaperone-assisted transfer reactions. Further, we provide evidence that [2Fe-2S]-Grx4 delivers clusters to multiple classes of Fe-S targets via direct ligand exchange in a process that is both dynamic and reversible. Global fits of cluster transfer kinetics support a model in which Grx4 outcompetes terminal target proteins for IscU-bound [2Fe-2S] clusters and functions as an intermediate cluster carrier. Overall, these studies demonstrate the power of chemically conjugated fluorophore reporters for unraveling mechanistic details of biological metal cofactor assembly and distribution networks.

The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation

PubMed Central

Mancone, Carmine; Regazzo, Giulia; Spagnuolo, Manuela; Alonzi, Tonino; Carlomosti, Fabrizio; Lucia, Maria Dell’Anna; Dell, Giulia 'Omo; Picardo, Mauro; Ciana, Paolo; Capogrossi, Maurizio C; Tripodi, Marco; Magenta, Alessandra; Giulia, Maria Rizzo; Gurtner, Aymone; Piaggio, Giulia

2017-01-01

Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y–dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferation. PMID:27793050

Dissecting Escherichia coli Outer Membrane Biogenesis Using Differential Proteomics

PubMed Central

Martorana, Alessandra M.; Motta, Sara; Di Silvestre, Dario; Falchi, Federica; Dehò, Gianni; Mauri, Pierluigi; Sperandeo, Paola; Polissi, Alessandra

2014-01-01

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality. PMID:24967819

Current and future molecular diagnostics for prion diseases.

PubMed

Lehto, Marty T; Peery, Harry E; Cashman, Neil R

2006-07-01

It is now widely held that the infectious agents underlying the transmissible spongiform encephalopathies are prions, which are primarily composed of a misfolded, protease-resistant isoform of the host prion protein. Untreatable prion disorders include some human diseases, such as Creutzfeldt-Jakob disease, and diseases of economically important animals, such as bovine spongiform encephalopathy (cattle) and chronic wasting disease (deer and elk). Detection and diagnosis of prion disease (and presymptomatic incubation) is contingent upon developing novel assays, which exploit properties uniquely possessed by this misfolded protein complex, rather than targeting an agent-specific nucleic acid. This review highlights some of the conventional and disruptive technologies developed to respond to this challenge.

Isolation and Purification of Complex II from Proteus Mirabilis Strain ATCC 29245

PubMed Central

Shabbiri, Khadija; Ahmad, Waqar; Syed, Quratulain; Adnan, Ahmad

2010-01-01

A respiratory complex was isolated from plasma membrane of pathogenic Proteus mirabilis strain ATCC 29245. It was identified as complex II consisting of succinate:quinone oxidoreductase (EC 1.3.5.1) containing single heme b. The complex II was purified by ion-exchange chromatography and gel filtration. The molecular weight of purified complex was 116.5 kDa and it was composed of three subunits with molecular weights of 19 kDa, 29 kDa and 68.5 kDa. The complex II contained 9.5 nmoles of cytochrome b per mg protein. Heme staining indicated that the 19 kDa subunit was cytochrome b. Its reduced form showed absorptions peaks at 557.0, 524.8 and 424.4 nm. The α-band was shifted from 557.0 nm to 556.8 nm in pyridine ferrohemochrome spectrum. The succinate: quinone oxidoreductase activity was found to be high in this microorganism. PMID:24031557

mTOR in Down syndrome: Role in Aß and tau neuropathology and transition to Alzheimer disease-like dementia.

PubMed

Di Domenico, Fabio; Tramutola, Antonella; Foppoli, Cesira; Head, Elizabeth; Perluigi, Marzia; Butterfield, D Allan

2018-01-01

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase involved in the regulation of protein synthesis and degradation, longevity and cytoskeletal formation. The mTOR pathway represents a key growth and survival pathway involved in several diseases such as cancer, obesity, cardiovascular disease and neurodegenerative diseases. Numerous studies linked the alterations of mTOR pathway to age-dependent cognitive decline, pathogenesis of Alzheimer disease (AD) and AD-like dementia in Down syndrome (DS). DS is the most frequent chromosomal abnormality that causes intellectual disability. The neuropathology of AD in DS is complex and involves impaired mitochondrial function, defects in neurogenesis, increased oxidative stress, altered proteostasis and autophagy networks as a result of triplication of chromosome 21(chr 21). The chr21 gene products are considered a principal neuropathogenic moiety in DS. Several genes involved respectively in the formation of senile plaques and neurofibrillary tangles (NFT), two main pathological hallmarks of AD, are mapped on chr21. Further, in subjects with DS the activation of mTOR signaling contributes to Aβ generation and the formation of NFT. This review discusses recent research highlighting the complex role of mTOR associated with the presence of two hallmarks of AD pathology, senile plaques (composed mostly of fibrillar Aß peptides), and NFT (composed mostly of hyperphosphorylated tau protein). Oxidative stress, associated with chr21-related Aβ and mitochondrial alterations, may significantly contribute to this linkage of mTOR to AD-like neuropathology in DS. Copyright © 2017 Elsevier Inc. All rights reserved.

Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx.

PubMed

Romano, Christine A; Zhou, Mowei; Song, Yang; Wysocki, Vicki H; Dohnalkova, Alice C; Kovarik, Libor; Paša-Tolić, Ljiljana; Tebo, Bradley M

2017-09-29

Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase in Bacillus sp. PL-12, Mnx, is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins, MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant to crystallization, so its structure is unknown. Here, we show that native mass spectrometry defines the subunit topology and copper binding of Mnx, while high-resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for understanding Mn biomineralization by such unexplored enzymes.Significant challenges exist for structural characterization of enzymes responsible for biomineralization. Here the authors show that native mass spectrometry and high resolution electron microscopy can define the subunit topology and copper binding of a manganese oxidizing complex, and describe early stage formation of its mineral products.

State of the APC/C: Organization, function, and structure

PubMed Central

McLean, Janel R.; Chaix, Denis; Ohi, Melanie D.; Gould, Kathleen L.

2016-01-01

The ubiquitin-proteasome protein degradation system is involved in many essential cellular processes including cell cycle regulation, cell differentiation, and the unfolded protein response.The anaphase-promoting complex/cyclosome (APC/C), an evolutionary conserved E3 ubiquitin ligase, was discovered 15 years ago because of its pivotal role in cyclin degradation and mitotic progression. Since then, we have learned that the APC/C is a very large, complex E3 ligase composed of 13 subunits, yielding a molecular machine of approximately 1 MDa. The intricate regulation of the APC/C is mediated by the Cdc20 family of activators, pseudosubstrate inhibitors, protein kinases and phosphatases and the spindle assembly checkpoint. The large size, complexity, and dynamic nature of the APC/C represent significant obstacles toward high-resolution structural techniques; however, over the last decade, there have been a number of lower resolution APC/C structures determined using single particle electron microscopy. These structures, when combined with data generated from numerous genetic and biochemical studies, have begun to shed light on how APC/C activity is regulated. Here, we discuss the most recent developments in the APC/C field concerning structure, substrate recognition, and catalysis. PMID:21261459

Structure of the heterotrimeric complex that regulates type III secretion needle formation

PubMed Central

Quinaud, Manuelle; Plé, Sophie; Job, Viviana; Contreras-Martel, Carlos; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa

2007-01-01

Type III secretion systems (T3SS), found in several Gram-negative pathogens, are nanomachines involved in the transport of virulence effectors directly into the cytoplasm of target cells. T3SS are essentially composed of basal membrane-embedded ring-like structures and a hollow needle formed by a single polymerized protein. Within the bacterial cytoplasm, the T3SS needle protein requires two distinct chaperones for stabilization before its secretion, without which the entire T3SS is nonfunctional. The 2.0-Å x-ray crystal structure of the PscE-PscF55–85-PscG heterotrimeric complex from Pseudomonas aeruginosa reveals that the C terminus of the needle protein PscF is engulfed within the hydrophobic groove of the tetratricopeptide-like molecule PscG, indicating that the macromolecular scaffold necessary to stabilize the T3SS needle is totally distinct from chaperoned complexes between pilus- or flagellum-forming molecules. Disruption of specific PscG–PscF interactions leads to impairment of bacterial cytotoxicity toward macrophages, indicating that this essential heterotrimer, which possesses homologs in a wide variety of pathogens, is a unique attractive target for the development of novel antibacterials. PMID:17470796

Structure and function of complex I in animals and plants - a comparative view.

PubMed

Senkler, Jennifer; Senkler, Michael; Braun, Hans-Peter

2017-09-01

The mitochondrial NADH dehydrogenase complex (complex I) has a molecular mass of about 1000 kDa and includes 40-50 subunits in animals, fungi and plants. It is composed of a membrane arm and a peripheral arm and has a conserved L-like shape in all species investigated. However, in plants and possibly some protists it has a second peripheral domain which is attached to the membrane arm on its matrix exposed side at a central position. The extra domain includes proteins resembling prokaryotic gamma-type carbonic anhydrases. We here present a detailed comparison of complex I from mammals and flowering plants. Forty homologous subunits are present in complex I of both groups of species. In addition, five subunits are present in mammalian complex I, which are absent in plants, and eight to nine subunits are present in plant complex I which do not occur in mammals. Based on the atomic structure of mammalian complex I and biochemical insights into complex I architecture from plants we mapped the species-specific subunits. Interestingly, four of the five animal-specific and five of the eight to nine plant-specific subunits are localized at the inner surface of the membrane arm of complex I in close proximity. We propose that the inner surface of the membrane arm represents a workbench for attaching proteins to complex I, which are not directly related to respiratory electron transport, like nucleoside kinases, acyl-carrier proteins or carbonic anhydrases. We speculate that further enzyme activities might be bound to this micro-location in other groups of organisms. © 2017 Scandinavian Plant Physiology Society.

Probing protein-lipid interactions by FRET between membrane fluorophores

NASA Astrophysics Data System (ADS)

Trusova, Valeriya M.; Gorbenko, Galyna P.; Deligeorgiev, Todor; Gadjev, Nikolai

2016-09-01

Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein-lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

RNA-dependent RNA polymerases of dsRNA bacteriophages.

PubMed

Makeyev, Eugene V; Grimes, Jonathan M

2004-04-01

Genome replication and transcription of riboviruses are catalyzed by an RNA-dependent RNA polymerase (RdRP). RdRPs are normally associated with other virus- or/and host-encoded proteins that modulate RNA polymerization activity and template specificity. The polymerase complex of double-stranded dsRNA viruses is a large icosahedral particle (inner core) containing RdRP as a minor constituent. In phi6 and other dsRNA bacteriophages from the Cystoviridae family, the inner core is composed of four virus-specific proteins. Of these, protein P2, or Pol subunit, has been tentatively identified as RdRP by sequence comparisons, but the role of this protein in viral RNA synthesis has not been studied until recently. Here, we overview the work on the Pol subunits of phi6 and related viruses from the standpoints of function, structure and evolution.

Examining protein-lipid interactions in model systems with a new squarylium fluorescent dye.

PubMed

Ioffe, Valeriya M; Gorbenko, Galyna P; Tatarets, Anatoliy L; Patsenker, Leonid D; Terpechnig, Ewald A

2006-07-01

The applicability of newly synthesized squarylium dye Sq to probing the changes in physical characteristics of lipid bilayer on the formation of protein-lipid complexes has been evaluated. Lipid vesicles composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with positively charged detergent cetyltrimethylammonium bromide (CTAB), anionic phospholipid cardiolipin (CL), and cholesterol (Chol) were employed as lipid component of model membrane systems while protein constituent was represented by lysozyme (Lz). Fluorescence intensity of Sq was found to decrease on Lz association with lipid bilayer. This effect was observed in all kinds of model systems suggesting that Sq is sensitive to modification of lipid bilayer physical properties on hydrophobic protein-lipid interactions. It was found that Sq spectral response to variations in Chol content depends on relative contributions of electrostatic and hydrophobic components of Lz-membrane binding.

Structure of a pantothenate transporter and implications for ECF module sharing and energy coupling of group II ECF transporters

PubMed Central

Zhang, Minhua; Bao, Zhihao; Zhao, Qin; Guo, Hui; Xu, Ke; Wang, Chengcheng

2014-01-01

Energy-coupling factor (ECF) transporters are a unique group of ATP-binding cassette (ABC) transporters responsible for micronutrient uptake from the environment. Each ECF transporter is composed of an S component (or EcfS protein) and T/A/A′ components (or EcfT/A/A′ proteins; ECF module). Among the group II ECF transporters, several EcfS proteins share one ECF module; however, the underlying mechanism remains unknown. Here we report the structure of a group II ECF transporter–pantothenate transporter from Lactobacillus brevis (LbECF-PanT), which shares the ECF module with the folate and hydroxymethylpyrimidine transporters (LbECF-FolT and LbECF-HmpT). Structural and mutational analyses revealed the residues constituting the pantothenate-binding pocket. We found that although the three EcfS proteins PanT, FolT, and HmpT are dissimilar in sequence, they share a common surface area composed of the transmembrane helices 1/2/6 (SM1/2/6) to interact with the coupling helices 2/3 (CH2/3) of the same EcfT. CH2 interacts mainly with SM1 via hydrophobic interactions, which may modulate the sliding movement of EcfS. CH3 binds to a hydrophobic surface groove formed by SM1, SM2, and SM6, which may transmit the conformational changes from EcfA/A′ to EcfS. We also found that the residues at the intermolecular surfaces in LbECF-PanT are essential for transporter activity, and that these residues may mediate intermolecular conformational transmission and/or affect transporter complex stability. In addition, we found that the structure of EcfT is conformationally dynamic, which supports its function as a scaffold to mediate the interaction of the ECF module with various EcfS proteins to form different transporter complexes. PMID:25512487

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting.

PubMed

Herbert, Kristina M; Sarkar, Susanta K; Mills, Maria; Delgado De la Herran, Hilda C; Neuman, Keir C; Steitz, Joan A

2016-02-01

During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem-loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins. © 2016 Herbert et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Lysosomal Multienzyme Complex: Pros and Cons of Working Together

PubMed Central

Bonten, Erik J.; Annunziata, Ida; d’Azzo, Alessandra

2014-01-01

The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, Protective Protein/Cathepsin A (PPCA), the sialidase, Neuraminidase-1 (NEU1), and the glycosidase β-Galactosidase (β-GAL). Next to this ‘core’ complex, the existence of sub-complexes, that may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1 and β-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders. PMID:24337808

Solution structure of the N-terminal domain of a replication restart primosome factor, PriC, in Escherichia coli

PubMed Central

Aramaki, Takahiko; Abe, Yoshito; Katayama, Tsutomu; Ueda, Tadashi

2013-01-01

In eubacterial organisms, the oriC-independent primosome plays an essential role in replication restart after the dissociation of the replication DNA-protein complex by DNA damage. PriC is a key protein component in the replication restart primosome. Our recent study suggested that PriC is divided into two domains: an N-terminal and a C-terminal domain. In the present study, we determined the solution structure of the N-terminal domain, whose structure and function have remained unknown until now. The revealed structure was composed of three helices and one extended loop. We also observed chemical shift changes in the heteronuclear NMR spectrum and oligomerization in the presence of ssDNA. These abilities may contribute to the PriC-ssDNA complex, which is important for the replication restart primosome. PMID:23868391

Detection of human Dicer and Argonaute 2 catalytic activity

PubMed Central

Perron, Marjorie P.; Landry, Patricia; Plante, Isabelle; Provost, Patrick

2013-01-01

The microRNA (miRNA)-guided RNA silencing pathway is a central and well-defined cellular process involved in messenger RNA (mRNA) translational control. This complex regulatory process is achieved by a well orchestrated machinery composed of a relatively few protein components, among which the ribonuclease III (RNase III) Dicer and Argonaute 2 (Ago2) play a central role. These two proteins are essential and it is of particular interest to measure and detect their catalytic activity under various situations and/or conditions. In this chapter, we describe different protocols that aim to study and determine the catalytic activity of Dicer and Ago2 in cell extracts, immune complexes and size-fractionated cell extracts. Another protocol aimed at assessing miRNA binding to Ago2 is also described. These experimental approaches are likely to be useful to researchers investigating the main steps of miRNA biogenesis and function in human health and diseases. PMID:21528451

Form follows function: the importance of endoplasmic reticulum shape.

PubMed

Westrate, L M; Lee, J E; Prinz, W A; Voeltz, G K

2015-01-01

The endoplasmic reticulum (ER) has a remarkably complex structure, composed of a single bilayer that forms the nuclear envelope, along with a network of sheets and dynamic tubules. Our understanding of the biological significance of the complex architecture of the ER has improved dramatically in the last few years. The identification of proteins and forces required for maintaining ER shape, as well as more advanced imaging techniques, has allowed the relationship between ER shape and function to come into focus. These studies have also revealed unexpected new functions of the ER and novel ER domains regulating alterations in ER dynamics. The importance of ER structure has become evident as recent research has identified diseases linked to mutations in ER-shaping proteins. In this review, we discuss what is known about the maintenance of ER architecture, the relationship between ER structure and function, and diseases associated with defects in ER structure.

The colloidal state of tannins impacts the nature of their interaction with proteins: the case of salivary proline-rich protein/procyanidins binding.

PubMed

Cala, Olivier; Dufourc, Erick J; Fouquet, Eric; Manigand, Claude; Laguerre, Michel; Pianet, Isabelle

2012-12-18

While the definition of tannins has been historically associated with its propensity to bind proteins in a nonspecific way, it is now admitted that specific interaction also occurs. The case of the astringency perception is a good example to illustrate this phenomenon: astringency is commonly described as a tactile sensation induced by the precipitation of a complex composed of proline-rich proteins present in the human saliva and tannins present in beverages such as tea or red wines. In the present work, the interactions between a human saliva protein segment and three different procyanidins (B1, B3, and C2) were investigated at the atomic level by NMR and molecular dynamics. The data provided evidence for (i) an increase in affinity compared to shortest human saliva peptides, which is accounted for by protein "wraping around" the tannin, (ii) a specificity in the interaction below tannin critical micelle concentration (CMC) of ca. 10 mM, with an affinity scale such that C2 > B1 > B3, and (iii) a nonspecific binding above tannin CMC that conducts irremediably to the precipitation of the tannins/protein complex. Such physicochemical findings describe in accurate terms saliva protein-tannin interactions and provide support for a more subtle description by oenologists of wine astringency perception in the mouth.

Bacterial expression and purification of a heterodimeric adrenomedullin receptor extracellular domain complex using DsbC-assisted disulfide shuffling.

PubMed

Hill, Heather E; Pioszak, Augen A

2013-03-01

Adrenomedullin (AM) is a peptide hormone that is a potent vasodilator and is essential for vascular development. The AM receptor is a heterodimeric cell surface receptor composed of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, in association with either of two receptor activity modifying protein (RAMP) coreceptors, RAMP2 or -3. The extracellular domains (ECDs) of CLR and the RAMPs form the primary AM binding site. Here, we present novel methodology for expression and purification of a heterodimeric AM receptor ECD complex as an MBP-CLR ECD fusion protein in association with the RAMP2 ECD. Co-expression of the RAMP2 ECD with the disulfide bond isomerase DsbC in the oxidizing cytoplasm of E. coli trxB gor enabled proper disulfide formation in vivo. The isolated RAMP2 ECD was purified to homogeneity. Co-expression of a soluble MBP-CLR ECD fusion protein with DsbC in E. coli trxB gor yielded a heterogeneous mixture of species with misfolded ECD. Incubation of affinity-purified MBP-CLR ECD in vitro with purified RAMP2 ECD, DsbC, and glutathione redox buffer promoted proper folding of the CLR ECD and formation of a stable MBP-CLR ECD:RAMP2 ECD complex that was purified by size-exclusion chromatography and which exhibited specific AM binding. Approximately 40mg of highly purified complex was obtained starting with 6L bacterial cultures for each protein. The methodology reported here will facilitate structure/function studies of the AM receptor. Copyright © 2012 Elsevier Inc. All rights reserved.

Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection.

PubMed

Albecka, Anna; Owen, Danielle J; Ivanova, Lyudmila; Brun, Juliane; Liman, Rukayya; Davies, Laura; Ahmed, M Firoz; Colaco, Susanna; Hollinshead, Michael; Graham, Stephen C; Crump, Colin M

2017-01-15

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells. Copyright © 2017 Albecka et al.

Interactions between the Nse3 and Nse4 Components of the SMC5-6 Complex Identify Evolutionarily Conserved Interactions between MAGE and EID Families

PubMed Central

Kozakova, Lucie; Liao, Chunyan; Guerineau, Marc; Colnaghi, Rita; Vidot, Susanne; Marek, Jaromir; Bathula, Sreenivas R.; Lehmann, Alan R.; Palecek, Jan

2011-01-01

Background The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6–8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors. Methodology/Principal Findings Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins. Conclusions/Significance We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins. PMID:21364888

The Nucleolar Fibrillarin Protein Is Required for Helper Virus-Independent Long-Distance Trafficking of a Subviral Satellite RNA in Plants[OPEN

PubMed Central

Chang, Chih-Hao; Lee, Shu-Chuan; Lo, Yih-Shan; Wang, Jiun-Da; Shaw, Jane; Chang, Ban-Yang

2016-01-01

RNA trafficking plays pivotal roles in regulating plant development, gene silencing, and adaptation to environmental stress. Satellite RNAs (satRNAs), parasites of viruses, depend on their helper viruses (HVs) for replication, encapsidation, and efficient spread. However, it remains largely unknown how satRNAs interact with viruses and the cellular machinery to undergo trafficking. Here, we show that the P20 protein of Bamboo mosaic potexvirus satRNA (satBaMV) can functionally complement in trans the systemic trafficking of P20-defective satBaMV in infected Nicotiana benthamiana. The transgene-derived satBaMV, uncoupled from HV replication, was able to move autonomously across a graft union identified by RT-qPCR, RNA gel blot, and in situ RT-PCR analyses. Coimmunoprecipitation experiments revealed that the major nucleolar protein fibrillarin is coprecipitated in the P20 protein complex. Notably, silencing fibrillarin suppressed satBaMV-, but not HV-, phloem-based movement following grafting or coinoculation with HV. Confocal microscopy revealed that the P20 protein colocalized with fibrillarin in the nucleoli and formed punctate structures associated with plasmodesmata. The mobile satBaMV RNA appears to exist as ribonucleoprotein (RNP) complex composed of P20 and fibrillarin, whereas BaMV movement proteins, capsid protein, and BaMV RNA are recruited with HV coinfection. Taken together, our findings provide insight into movement of satBaMV via the fibrillarin-satBaMV-P20 RNP complex in phloem-mediated systemic trafficking. PMID:27702772

Cyclophilins facilitate dissociation of the human papillomavirus type 16 capsid protein L1 from the L2/DNA complex following virus entry.

PubMed

Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L; Sapp, Martin

2012-09-01

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.

Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry

PubMed Central

Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L.

2012-01-01

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles. PMID:22761365

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

PubMed

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

A recruiting protein of geranylgeranyl diphosphate synthase controls metabolic flux toward chlorophyll biosynthesis in rice.

PubMed

Zhou, Fei; Wang, Cheng-Yuan; Gutensohn, Michael; Jiang, Ling; Zhang, Peng; Zhang, Dabing; Dudareva, Natalia; Lu, Shan

2017-06-27

In plants, geranylgeranyl diphosphate (GGPP) is produced by plastidic GGPP synthase (GGPPS) and serves as a precursor for vital metabolic branches, including chlorophyll, carotenoid, and gibberellin biosynthesis. However, molecular mechanisms regulating GGPP allocation among these biosynthetic pathways localized in the same subcellular compartment are largely unknown. We found that rice contains only one functionally active GGPPS, OsGGPPS1, in chloroplasts. A functionally active homodimeric enzyme composed of two OsGGPPS1 subunits is located in the stroma. In thylakoid membranes, however, the GGPPS activity resides in a heterodimeric enzyme composed of one OsGGPPS1 subunit and GGPPS recruiting protein (OsGRP). OsGRP is structurally most similar to members of the geranyl diphosphate synthase small subunit type II subfamily. In contrast to members of this subfamily, OsGRP enhances OsGGPPS1 catalytic efficiency and specificity of GGPP production on interaction with OsGGPPS1. Structural biology and protein interaction analyses demonstrate that affinity between OsGRP and OsGGPPS1 is stronger than between two OsGGPPS1 molecules in homodimers. OsGRP determines OsGGPPS1 suborganellar localization and directs it to a large protein complex in thylakoid membranes, consisting of geranylgeranyl reductase (OsGGR), light-harvesting-like protein 3 (OsLIL3), protochlorophyllide oxidoreductase (OsPORB), and chlorophyll synthase (OsCHLG). Taken together, genetic and biochemical analyses suggest OsGRP functions in recruiting OsGGPPS1 from the stroma toward thylakoid membranes, thus providing a mechanism to control GGPP flux toward chlorophyll biosynthesis.

Deciphering complex dynamics of water counteraction around secondary structural elements of allosteric protein complex: Case study of SAP-SLAM system in signal transduction cascade

NASA Astrophysics Data System (ADS)

Samanta, Sudipta; Mukherjee, Sanchita

2018-01-01

The first hydration shell of a protein exhibits heterogeneous behavior owing to several attributes, majorly local polarity and structural flexibility as revealed by solvation dynamics of secondary structural elements. We attempt to recognize the change in complex water counteraction generated due to substantial alteration in flexibility during protein complex formation. The investigation is carried out with the signaling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, and interacting with SLAM-associated protein (SAP), composed of one SH2 domain. All atom molecular dynamics simulations are employed to the aqueous solutions of free SAP and SLAM-peptide bound SAP. We observed that water dynamics around different secondary structural elements became highly affected as well as nicely correlated with the SLAM-peptide induced change in structural rigidity obtained by thermodynamic quantification. A few instances of contradictory dynamic features of water to the change in structural flexibility are explained by means of occluded polar residues by the peptide. For βD, EFloop, and BGloop, both structural flexibility and solvent accessibility of the residues confirm the obvious contribution. Most importantly, we have quantified enhanced restriction in water dynamics around the second Fyn-binding site of the SAP due to SAP-SLAM complexation, even prior to the presence of Fyn. This observation leads to a novel argument that SLAM induced more restricted water molecules could offer more water entropic contribution during the subsequent Fyn binding and provide enhanced stability to the SAP-Fyn complex in the signaling cascade. Finally, SLAM induced water counteraction around the second binding site of the SAP sheds light on the allosteric property of the SAP, which becomes an integral part of the underlying signal transduction mechanism.

Deciphering complex dynamics of water counteraction around secondary structural elements of allosteric protein complex: Case study of SAP-SLAM system in signal transduction cascade.

PubMed

Samanta, Sudipta; Mukherjee, Sanchita

2018-01-28

The first hydration shell of a protein exhibits heterogeneous behavior owing to several attributes, majorly local polarity and structural flexibility as revealed by solvation dynamics of secondary structural elements. We attempt to recognize the change in complex water counteraction generated due to substantial alteration in flexibility during protein complex formation. The investigation is carried out with the signaling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, and interacting with SLAM-associated protein (SAP), composed of one SH2 domain. All atom molecular dynamics simulations are employed to the aqueous solutions of free SAP and SLAM-peptide bound SAP. We observed that water dynamics around different secondary structural elements became highly affected as well as nicely correlated with the SLAM-peptide induced change in structural rigidity obtained by thermodynamic quantification. A few instances of contradictory dynamic features of water to the change in structural flexibility are explained by means of occluded polar residues by the peptide. For βD, EFloop, and BGloop, both structural flexibility and solvent accessibility of the residues confirm the obvious contribution. Most importantly, we have quantified enhanced restriction in water dynamics around the second Fyn-binding site of the SAP due to SAP-SLAM complexation, even prior to the presence of Fyn. This observation leads to a novel argument that SLAM induced more restricted water molecules could offer more water entropic contribution during the subsequent Fyn binding and provide enhanced stability to the SAP-Fyn complex in the signaling cascade. Finally, SLAM induced water counteraction around the second binding site of the SAP sheds light on the allosteric property of the SAP, which becomes an integral part of the underlying signal transduction mechanism.

Native structure of a type IV secretion system core complex essential for Legionella pathogenesis.

PubMed

Kubori, Tomoko; Koike, Masafumi; Bui, Xuan Thanh; Higaki, Saori; Aizawa, Shin-Ichi; Nagai, Hiroki

2014-08-12

Bacterial type IV secretion systems are evolutionarily related to conjugation systems and play a pivotal role in infection by delivering numerous virulence factors into host cells. Using transmission electron microscopy, we report the native molecular structure of the core complex of the Dot/Icm type IV secretion system encoded by Legionella pneumophila, an intracellular human pathogen. The biochemically isolated core complex, composed of at least five proteins--DotC, DotD, DotF, DotG, and DotH--has a ring-shaped structure. Intriguingly, morphologically distinct premature complexes are formed in the absence of DotG or DotF. Our data suggest that DotG forms a central channel spanning inner and outer membranes. DotF, a component dispensable for type IV secretion, plays a role in efficient embedment of DotG into the functional core complex. These results highlight a common scheme for the biogenesis of transport machinery.

The devil is in the details: comparison between COP9 signalosome (CSN) and the LID of the 26S proteasome.

PubMed

Meister, Cindy; Gulko, Miriam Kolog; Köhler, Anna M; Braus, Gerhard H

2016-02-01

The COP9 signalosome (CSN) and the proteasomal LID are conserved macromolecular complexes composed of at least eight subunits with molecular weights of approximately 350 kDa. CSN and LID are part of the ubiquitin–proteasome pathway and cleave isopeptide linkages of lysine side chains on target proteins. CSN cleaves the isopeptide bond of ubiquitin-like protein Nedd8 from cullins, whereas the LID cleaves ubiquitin from target proteins sentenced for degradation. CSN and LID are structurally and functionally similar but the order of the assembly pathway seems to be different. The assembly differs in at least the last subunit joining the pre-assembled subcomplex. This review addresses the similarities and differences in structure, function and assembly of CSN and LID.

Top-Down Protein Identification of Proteasome Proteins with nanoLC FT-ICR MS Employing Data-Independent Fragmentation Methods

PubMed Central

Lakshmanan, Rajeswari; Wolff, Jeremy J.; Alvarado, Rudy; Loo, Joseph A.

2014-01-01

A comparison of different data-independent fragmentation methods combined with liquid chromatography (LC) coupled to high resolution Fourier-transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS) is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complex and their post-translational modifications were identified using a 15-Tesla FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty cycle measurements that better suit on-line LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (Continuous Accumulation of Selected Ions)-CAD. The N-terminus for 9 out of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass measurement accuracy with the LC-FT-ICR system for the 20–30 kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100 kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact protein fragmentation and is an effective addition to the growing inventory of dissociation methods which are compatible with on-line protein separation coupled to FT-ICR MS. PMID:24478249

Manganese-dependent carboanhydrase activity of photosystem II proteins.

PubMed

Shitov, A V; Pobeguts, O V; Smolova, T N; Allakhverdiev, S I; Klimov, V V

2009-05-01

Four sources of carbonic anhydrase (CA) activity in submembrane preparations of photosystem II (PS II) isolated from pea leaves were examined. Three of them belong to the hydrophilic proteins of the oxygen-evolving complex of PS II with molecular mass 33 kDa (protein PsbO), 24 kDa (protein PsbP), and 18 kDa (protein PsbQ). The fourth source of CA activity is associated with a pigment-protein complex of PS II after removing three hydrophilic proteins by salt treatment. Except for protein PsbQ, the CA activity of all these proteins depends on the presence of Mn2+: the purified protein PsbO did not show CA activity before adding Mn2+ into the medium (concentration of Mn2+ required for 50% effect, EC(50), was 670 microM); CA activity of protein mixture composed of PsbP and PsbQ increased more than 5-fold upon adding Mn2+ (EC(50) was 45 microM). CA activity of purified protein PsbP increased 2-fold in the presence of 200 microM Mn2+. As indicated for the mixture of two proteins (PsbP and PsbQ), Mg2+, Ca2+, and Zn2+, in contrast to Mn2+, suppressed CA activity (both initial and Mn2+-induced activity). Since the found sources of CA activity demonstrated properties different from ones of typical CA (need for Mn2+, insensitivity or low sensitivity to acetazolamide or ethoxyzolamide) and such CA activity was found only among PS II proteins, we cannot exclude that they belong to the type of Mn-dependent CA associated with PS II.

PepComposer: computational design of peptides binding to a given protein surface

PubMed Central

Obarska-Kosinska, Agnieszka; Iacoangeli, Alfredo; Lepore, Rosalba; Tramontano, Anna

2016-01-01

There is a wide interest in designing peptides able to bind to a specific region of a protein with the aim of interfering with a known interaction or as starting point for the design of inhibitors. Here we describe PepComposer, a new pipeline for the computational design of peptides binding to a given protein surface. PepComposer only requires the target protein structure and an approximate definition of the binding site as input. We first retrieve a set of peptide backbone scaffolds from monomeric proteins that harbor the same backbone arrangement as the binding site of the protein of interest. Next, we design optimal sequences for the identified peptide scaffolds. The method is fully automatic and available as a web server at http://biocomputing.it/pepcomposer/webserver. PMID:27131789

Purification and characterisation of the fission yeast Ndc80 complex.

PubMed

Matsuo, Yuzy; Maurer, Sebastian P; Surrey, Thomas; Toda, Takashi

2017-07-01

The Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that multiple microtubule-associated proteins including the Dis1/chTOG microtubule polymerase and the Mal3/EB1 microtubule plus-end tracking protein directly or indirectly bind Ndc80, thereby ensuring stable kinetochore-microtubule attachment. However, the purification of the Ndc80 complex from this yeast has not been achieved, which hampers the in-depth investigation as to how the outer kinetochore attaches to the plus end of the spindle microtubule. Here we report the two-step purification of the fission yeast Ndc80 holo complex from bacteria. First, we purified separately two sub-complexes consisting of Ndc80-Nuf2 and Spc24-Spc25. Then, these two sub-complexes were mixed and applied to size-exclusion chromatography. The reconstituted Ndc80 holo complex is composed of four subunits with equal stoichiometry. The complex possesses microtubule-binding activity, and Total Internal Reflection Fluorescence (TIRF)-microscopy assays show that the complex binds the microtubule lattice. Interestingly, unlike the human complex, the fission yeast complex does not track depolymerising microtubule ends. Further analysis shows that under physiological ionic conditions, the Ndc80 holo complex does not detectably bind Dis1, but instead it interacts with Mal3/EB1, by which the Ndc80 complex tracks the growing microtubule plus end. This result substantiates the notion that the Ndc80 complex plays a crucial role in establishment of the dynamic kinetochore-microtubule interface by cooperating with chTOG and EB1. Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

Structure of faustovirus, a large dsDNA virus

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

Klose, Thomas; Reteno, Dorine G.; Benamar, Samia

Many viruses protect their genome with a combination of a protein shell with or without a membrane layer. In this paper, we describe the structure of faustovirus, the first DNA virus (to our knowledge) that has been found to use two protein shells to encapsidate and protect its genome. The crystal structure of the major capsid protein, in combination with cryo-electron microscopy structures of two different maturation stages of the virus, shows that the outer virus shell is composed of a double jelly-roll protein that can be found in many double-stranded DNA viruses. The structure of the repeating hexameric unitmore » of the inner shell is different from all other known capsid proteins. In addition to the unique architecture, the region of the genome that encodes the major capsid protein stretches over 17,000 bp and contains a large number of introns and exons. Finally, this complexity might help the virus to rapidly adapt to new environments or hosts.« less

Structure of faustovirus, a large dsDNA virus

DOE PAGES

Klose, Thomas; Reteno, Dorine G.; Benamar, Samia; ...

2016-05-16

Many viruses protect their genome with a combination of a protein shell with or without a membrane layer. In this paper, we describe the structure of faustovirus, the first DNA virus (to our knowledge) that has been found to use two protein shells to encapsidate and protect its genome. The crystal structure of the major capsid protein, in combination with cryo-electron microscopy structures of two different maturation stages of the virus, shows that the outer virus shell is composed of a double jelly-roll protein that can be found in many double-stranded DNA viruses. The structure of the repeating hexameric unitmore » of the inner shell is different from all other known capsid proteins. In addition to the unique architecture, the region of the genome that encodes the major capsid protein stretches over 17,000 bp and contains a large number of introns and exons. Finally, this complexity might help the virus to rapidly adapt to new environments or hosts.« less

Classification of ligand molecules in PDB with fast heuristic graph match algorithm COMPLIG.

PubMed

Saito, Mihoko; Takemura, Naomi; Shirai, Tsuyoshi

2012-12-14

A fast heuristic graph-matching algorithm, COMPLIG, was devised to classify the small-molecule ligands in the Protein Data Bank (PDB), which are currently not properly classified on structure basis. By concurrently classifying proteins and ligands, we determined the most appropriate parameter for categorizing ligands to be more than 60% identity of atoms and bonds between molecules, and we classified 11,585 types of ligands into 1946 clusters. Although the large clusters were composed of nucleotides or amino acids, a significant presence of drug compounds was also observed. Application of the system to classify the natural ligand status of human proteins in the current database suggested that, at most, 37% of the experimental structures of human proteins were in complex with natural ligands. However, protein homology- and/or ligand similarity-based modeling was implied to provide models of natural interactions for an additional 28% of the total, which might be used to increase the knowledge of intrinsic protein-metabolite interactions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: capture of and report on drug and protein payloads.

PubMed

Wong, Chin Ken; Laos, Alistair J; Soeriyadi, Alexander H; Wiedenmann, Jörg; Curmi, Paul M G; Gooding, J Justin; Marquis, Christopher P; Stenzel, Martina H; Thordarson, Pall

2015-04-27

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Interaction of Streptococcal Enolase with Canine Plasminogen: The Role of Surfaces in Complex Formation

PubMed Central

Balhara, Vinod; Deshmukh, Sasmit S.; Kálmán, László; Kornblatt, Jack A.

2014-01-01

The enolase from Streptococcus pyogenes (Str enolase F137L/E363G) is a homo-octamer shaped like a donut. Plasminogen (Pgn) is a monomeric protein composed of seven discrete separated domains organized into a lock washer. The enolase is known to bind Pgn. In past work we searched for conditions in which the two proteins would bind to one another. The two native proteins in solution would not bind under any of the tried conditions. We found that if the structures were perturbed binding would occur. We stated that only the non-native Str enolase or Pgn would interact such that we could detect binding. We report here the results of a series of dual polarization interferometry (DPI) experiments coupled with atomic force microscopy (AFM), isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence. We show that the critical condition for forming stable complexes of the two native proteins involves Str enolase binding to a surface. Surfaces that attract Str enolase are a sufficient condition for binding Pgn. Under certain conditions, Pgn adsorbed to a surface will bind Str enolase. PMID:24520380

Vps26B-retromer negatively regulates plasma membrane resensitization of PAR-2.

PubMed

Bugarcic, Andrea; Vetter, Irina; Chalmers, Silke; Kinna, Genevieve; Collins, Brett M; Teasdale, Rohan D

2015-11-01

Retromer is a trimeric complex composed of Vps26, Vps29, and Vps35 and has been shown to be involved in trafficking and sorting of transmembrane proteins within the endosome. The Vps26 paralog, Vps26B, defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. Although endosomally associated, Vps26B-retromer does not bind the established retromer transmembrane cargo protein, cation-independent mannose 6-phosphate receptor (CI-M6PR), indicating it has a distinct role to retromer containing the Vps26A paralog. In the present study we use the previously established Vps26B-expressing HEK293 cell model to address the role of Vps26B-retromer in trafficking of the protease activated G-protein coupled receptor PAR-2 to the plasma membrane. In these cells there is no apparent defect in the initial activation of the receptor, as evidenced by release of intracellular calcium, ERK1/2 signaling and endocytosis of activated receptor PAR-2 into degradative organelles. However, we observe a significant delay in plasma membrane repopulation of the protease activated G protein-coupled receptor PAR-2 following stimulation, resulting in a defect in PAR-2 activation after resensitization. Here we propose that PAR-2 plasma membrane repopulation is regulated by Vps26B-retromer, describing a potential novel role for this complex. © 2015 International Federation for Cell Biology.

Self-Chaperoning of the Type III Secretion System needle tip proteins IpaD and BipD

PubMed Central

Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E.; Birket, Susan; Field, Terry; Picking, William D.; Blocker, Ariel; Galyov, Edouard E.; Picking, Wendy L.; Lea, Susan M.

2007-01-01

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence; IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intra-molecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure. PMID:17077085

Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD.

PubMed

Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E; Birket, Susan; Field, Terry; Picking, William D; Blocker, Ariel J; Galyov, Edouard E; Picking, Wendy L; Lea, Susan M

2007-02-09

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.

Structural Isosteres of Phosphate Groups in the Protein Data Bank.

PubMed

Zhang, Yuezhou; Borrel, Alexandre; Ghemtio, Leo; Regad, Leslie; Boije Af Gennäs, Gustav; Camproux, Anne-Claude; Yli-Kauhaluoma, Jari; Xhaard, Henri

2017-03-27

We developed a computational workflow to mine the Protein Data Bank for isosteric replacements that exist in different binding site environments but have not necessarily been identified and exploited in compound design. Taking phosphate groups as examples, the workflow was used to construct 157 data sets, each composed of a reference protein complexed with AMP, ADP, ATP, or pyrophosphate as well other ligands. Phosphate binding sites appear to have a high hydration content and large size, resulting in U-shaped bioactive conformations recurrently found across unrelated protein families. A total of 16 413 replacements were extracted, filtered for a significant structural overlap on phosphate groups, and sorted according to their SMILES codes. In addition to the classical isosteres of phosphate, such as carboxylate, sulfone, or sulfonamide, unexpected replacements that do not conserve charge or polarity, such as aryl, aliphatic, or positively charged groups, were found.

N-cadherin in adult rat cardiomyocytes in culture. II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes.

PubMed

Hertig, C M; Butz, S; Koch, S; Eppenberger-Eberhardt, M; Kemler, R; Eppenberger, H M

1996-01-01

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The 'redifferentiation model' of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin which bind to either beta-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the following gap junction formation; a temporal sequence of the appearance of adherens junction proteins and of gap junctions forming connexin-43 is suggested.

Low-Cost Synthesis of Smart Biocompatible Graphene Oxide Reduced Species by Means of GFP.

PubMed

Masullo, Tiziana; Armata, Nerina; Pendolino, Flavio; Colombo, Paolo; Lo Celso, Fabrizio; Mazzola, Salvatore; Cuttitta, Angela

2016-02-01

The aim of this work is focused on the engineering of biocompatible complex systems composed of an inorganic and bio part. Graphene oxide (GO) and/or graphite oxide (GtO) were taken into account as potential substrates to the linkage of the protein such as Anemonia sulcata recombinant green fluorescent protein (rAsGFP). The complex system is obtained through a reduction process between GO/GtO and rAsGFP archiving an environmentally friendly biosynthesis. Spectroscopic measurements support the formation of reduced species. In particular, photoluminescence shows a change in the activity of the protein when a bond is formed, highlighted by a loss of the maximum emission signal of rAsGFP and a redshift of the maximum absorption peak of the GO/GtO species. Moreover, the hemolysis assay reveals a lower value in the presence of less oxidized graphene species providing evidence for a biocompatible material. This singular aspect can be approached as a promising method for circulating pharmaceutical preparations via intravenous administration in the field of drug delivery.

Molecular Architecture of Full-length TRF1 Favors Its Interaction with DNA.

PubMed

Boskovic, Jasminka; Martinez-Gago, Jaime; Mendez-Pertuz, Marinela; Buscato, Alberto; Martinez-Torrecuadrada, Jorge Luis; Blasco, Maria A

2016-10-07

Telomeres are specific DNA-protein structures found at both ends of eukaryotic chromosomes that protect the genome from degradation and from being recognized as double-stranded breaks. In vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence that are bound by a six-subunit complex called shelterin. Molecular mechanisms of telomere functions remain unknown in large part due to lack of structural data on shelterins, shelterin complex, and its interaction with the telomeric DNA repeats. TRF1 is one of the best studied shelterin components; however, the molecular architecture of the full-length protein remains unknown. We have used single-particle electron microscopy to elucidate the structure of TRF1 and its interaction with telomeric DNA sequence. Our results demonstrate that full-length TRF1 presents a molecular architecture that assists its interaction with telometic DNA and at the same time makes TRFH domains accessible to other TRF1 binding partners. Furthermore, our studies suggest hypothetical models on how other proteins as TIN2 and tankyrase contribute to regulate TRF1 function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Architecture of Full-length TRF1 Favors Its Interaction with DNA*

PubMed Central

Boskovic, Jasminka; Martinez-Gago, Jaime; Mendez-Pertuz, Marinela; Buscato, Alberto; Martinez-Torrecuadrada, Jorge Luis; Blasco, Maria A.

2016-01-01

Telomeres are specific DNA-protein structures found at both ends of eukaryotic chromosomes that protect the genome from degradation and from being recognized as double-stranded breaks. In vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence that are bound by a six-subunit complex called shelterin. Molecular mechanisms of telomere functions remain unknown in large part due to lack of structural data on shelterins, shelterin complex, and its interaction with the telomeric DNA repeats. TRF1 is one of the best studied shelterin components; however, the molecular architecture of the full-length protein remains unknown. We have used single-particle electron microscopy to elucidate the structure of TRF1 and its interaction with telomeric DNA sequence. Our results demonstrate that full-length TRF1 presents a molecular architecture that assists its interaction with telometic DNA and at the same time makes TRFH domains accessible to other TRF1 binding partners. Furthermore, our studies suggest hypothetical models on how other proteins as TIN2 and tankyrase contribute to regulate TRF1 function. PMID:27563064

Molecular Architecture of Plant Thylakoids under Physiological and Light Stress Conditions: A Study of Lipid–Light-Harvesting Complex II Model Membranes[C][W

PubMed Central

Janik, Ewa; Bednarska, Joanna; Zubik, Monika; Puzio, Michal; Luchowski, Rafal; Grudzinski, Wojciech; Mazur, Radoslaw; Garstka, Maciej; Maksymiec, Waldemar; Kulik, Andrzej; Dietler, Giovanni; Gruszecki, Wieslaw I.

2013-01-01

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation. PMID:23898030

Molecular architecture of plant thylakoids under physiological and light stress conditions: a study of lipid-light-harvesting complex II model membranes.

PubMed

Janik, Ewa; Bednarska, Joanna; Zubik, Monika; Puzio, Michal; Luchowski, Rafal; Grudzinski, Wojciech; Mazur, Radoslaw; Garstka, Maciej; Maksymiec, Waldemar; Kulik, Andrzej; Dietler, Giovanni; Gruszecki, Wieslaw I

2013-06-01

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.

Deletion of PDZD7 disrupts the Usher syndrome type 2 protein complex in cochlear hair cells and causes hearing loss in mice.

PubMed

Zou, Junhuang; Zheng, Tihua; Ren, Chongyu; Askew, Charles; Liu, Xiao-Ping; Pan, Bifeng; Holt, Jeffrey R; Wang, Yong; Yang, Jun

2014-05-01

Usher syndrome type 2 (USH2) is the predominant form of USH, a leading genetic cause of combined deafness and blindness. PDZD7, a paralog of two USH causative genes, USH1C and USH2D (WHRN), was recently reported to be implicated in USH2 and non-syndromic deafness. It encodes a protein with multiple PDZ domains. To understand the biological function of PDZD7 and the pathogenic mechanism caused by PDZD7 mutations, we generated and thoroughly characterized a Pdzd7 knockout mouse model. The Pdzd7 knockout mice exhibit congenital profound deafness, as assessed by auditory brainstem response, distortion product otoacoustic emission and cochlear microphonics tests, and normal vestibular function, as assessed by their behaviors. Lack of PDZD7 leads to the disorganization of stereocilia bundles and a reduction in mechanotransduction currents and sensitivity in cochlear outer hair cells. At the molecular level, PDZD7 determines the localization of the USH2 protein complex, composed of USH2A, GPR98 and WHRN, to ankle links in developing cochlear hair cells, likely through its direct interactions with these three proteins. The localization of PDZD7 to the ankle links of cochlear hair bundles also relies on USH2 proteins. In photoreceptors of Pdzd7 knockout mice, the three USH2 proteins largely remain unchanged at the periciliary membrane complex. The electroretinogram responses of both rod and cone photoreceptors are normal in knockout mice at 1 month of age. Therefore, although the organization of the USH2 complex appears different in photoreceptors, it is clear that PDZD7 plays an essential role in organizing the USH2 complex at ankle links in developing cochlear hair cells. GenBank accession numbers: KF041446, KF041447, KF041448, KF041449, KF041450, KF041451.

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

PubMed Central

Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

1996-01-01

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 Fig. 7 Fig. 8 PMID:8632983

The consequences of the intracellular retention of pathogen-derived T-cell-independent antigens on protein presentation to T cells.

PubMed

Leyva-Cobián, F; Outschoorn, I M; Carrasco-Marín, E; Alvarez-Domínguez, C

1997-10-01

Intracellular pathogens can be considered as particulate antigens chemically composed of a complex mixture of T-cell-dependent antigens (TD) (peptides and proteins) and T-cell-independent antigens (TI) (glycolipids and complex polysaccharides). A large range of saccharides (from oligosaccharides to complex polysaccharides) derived from pathogenic microorganisms are being isolated and characterized. They are currently implicated in signaling systems and concomitant host-parasite relationships. However, there are not many structure-function relationships described for these pathogens. This is particularly true of polysaccharides. In this report we have reviewed the role of defined TI antigens in the processing and presentation of defined TD antigens to specific T cells by antigen-presenting cells (APC). We also considered the importance of some of the chemical characteristics shared by different carbohydrates implicated in the inhibition of antigen presentation. These findings are discussed in relation to the clear immunopathological consequences of long retention periods of complex carbohydrate molecules derived from intracellular parasites inside certain APC and the absence of antigen presentation impairment in physiological situations such as the removal of senescent or damaged red blood cells by splenic macrophages or intracellular accumulation of carbohydrates in colostrum and milk macrophages during lactation.

Functional analysis of dengue virus (DENV) type 2 envelope protein domain 3 type-specific and DENV complex-reactive critical epitope residues.

PubMed

Pitcher, Trevor J; Sarathy, Vanessa V; Matsui, Kiyohiko; Gromowski, Gregory D; Huang, Claire Y-H; Barrett, Alan D T

2015-02-01

The dengue virus (DENV) envelope protein domain 3 (ED3) is the target of potent virus neutralizing antibodies. The DENV-2 ED3 contains adjacent type-specific and DENV complex-reactive antigenic sites that are composed of a small number of residues that were previously demonstrated to be critical for antibody binding. Site-directed mutagenesis of a DENV-2 16681 infectious clone was used to mutate critical residues in the DENV-2 type-specific (K305A and P384A) and DENV complex-reactive (K310A) antigenic sites. The K305A mutant virus multiplied like the parent virus in mosquito and mammalian cells, as did the P384A mutant virus, which required a compensatory mutation (G330D) for viability. However, the K310A mutant virus could not be recovered. The DENV-2 type-specific critical residue mutations K305A and P384A+G330D reduced the ability of DENV-2 type-specific, but not DENV complex-reactive, mAbs to neutralize virus infectivity and this was directly correlated with mAb binding affinity to the rED3 mutants. © 2015 The Authors.

Crystallization of Macromolecules

PubMed Central

Friedmann, David; Messick, Troy; Marmorstein, Ronen

2014-01-01

X-ray crystallography has evolved into a very powerful tool to determine the three-dimensional structure of macromolecules and macromolecular complexes. The major bottleneck in structure determination by X-ray crystallography is the preparation of suitable crystalline samples. This unit outlines steps for the crystallization of a macromolecule, starting with a purified, homogeneous sample. The first protocols describe preparation of the macromolecular sample (i.e., proteins, nucleic acids, and macromolecular complexes). The preparation and assessment of crystallization trials is then described, along with a protocol for confirming whether the crystals obtained are composed of macromolecule as opposed to a crystallization reagent . Next, the optimization of crystallization conditions is presented. Finally, protocols that facilitate the growth of larger crystals through seeding are described. PMID:22045560

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

PubMed Central

Thorgersen, Michael P.; Lancaster, W. Andrew; Rajeev, Lara; Ge, Xiaoxuan; Vaccaro, Brian J.; Poole, Farris L.; Arkin, Adam P.; Mukhopadhyay, Aindrila

2016-01-01

ABSTRACT Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. IMPORTANCE Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Herein, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. While other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex. The UBC provides a potential tool for the microbiological sequestration of uranium for the cleaning of contaminated environments. PMID:27913415

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

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

Thorgersen, Michael P.; Lancaster, W. Andrew; Rajeev, Lara

Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC hadmore » an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Here in this paper, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. Finally, while other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex. The UBC provides a potential tool for the microbiological sequestration of uranium for the cleaning of contaminated environments.« less

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium.

PubMed

Thorgersen, Michael P; Lancaster, W Andrew; Rajeev, Lara; Ge, Xiaoxuan; Vaccaro, Brian J; Poole, Farris L; Arkin, Adam P; Mukhopadhyay, Aindrila; Adams, Michael W W

2017-02-15

Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Herein, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. While other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex. The UBC provides a potential tool for the microbiological sequestration of uranium for the cleaning of contaminated environments. Copyright © 2017 American Society for Microbiology.

The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

PubMed

Maurizy, Chloé; Quinternet, Marc; Abel, Yoann; Verheggen, Céline; Santo, Paulo E; Bourguet, Maxime; C F Paiva, Ana; Bragantini, Benoît; Chagot, Marie-Eve; Robert, Marie-Cécile; Abeza, Claire; Fabre, Philippe; Fort, Philippe; Vandermoere, Franck; M F Sousa, Pedro; Rain, Jean-Christophe; Charpentier, Bruno; Cianférani, Sarah; Bandeiras, Tiago M; Pradet-Balade, Bérengère; Manival, Xavier; Bertrand, Edouard

2018-05-29

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis.

Comparative analysis of the ternary complex factors Elk-1, SAP-1a and SAP-2 (ERP/NET).

PubMed Central

Price, M A; Rogers, A E; Treisman, R

1995-01-01

A transcription factor ternary complex composed of Serum Response Factor (SRF) and Ternary Complex Factor (TCF) mediates the response of the c-fos Serum Response Element (SRE) to growth factors and mitogens. Three Ets domain proteins, Elk-1, SAP-1 and ERP/NET, have been reported to have the properties of TCF. Here we compare Elk-1 and SAP-1a with the human ERP/NET homologue SAP-2. All three TCF RNAs are ubiquitously expressed at similar relative levels. All three proteins contain conserved regions that interact with SRF and the c-fos SRE with comparable efficiency, but in vitro complex formation by SAP-2 is strongly inhibited by its C-terminal sequences. Similarly, only Elk-1 and SAP-1a efficiently bind the c-fos SRE in vivo; ternary complex formation by SAP-2 is weak and is substantially unaffected by serum stimulation or v-ras co-expression. All three TCFs contain C-terminal transcriptional activation domains that are phosphorylated following growth factor stimulation. Activation requires conserved S/T-P motifs found in all the TCF family members. Each TCF activation domain can be phosphorylated in vitro by partially purified ERK2, and ERK activation in vivo is sufficient to potentiate transcriptional activation. Images PMID:7540136

Comparative analysis of the ternary complex factors Elk-1, SAP-1a and SAP-2 (ERP/NET).

PubMed

Price, M A; Rogers, A E; Treisman, R

1995-06-01

A transcription factor ternary complex composed of Serum Response Factor (SRF) and Ternary Complex Factor (TCF) mediates the response of the c-fos Serum Response Element (SRE) to growth factors and mitogens. Three Ets domain proteins, Elk-1, SAP-1 and ERP/NET, have been reported to have the properties of TCF. Here we compare Elk-1 and SAP-1a with the human ERP/NET homologue SAP-2. All three TCF RNAs are ubiquitously expressed at similar relative levels. All three proteins contain conserved regions that interact with SRF and the c-fos SRE with comparable efficiency, but in vitro complex formation by SAP-2 is strongly inhibited by its C-terminal sequences. Similarly, only Elk-1 and SAP-1a efficiently bind the c-fos SRE in vivo; ternary complex formation by SAP-2 is weak and is substantially unaffected by serum stimulation or v-ras co-expression. All three TCFs contain C-terminal transcriptional activation domains that are phosphorylated following growth factor stimulation. Activation requires conserved S/T-P motifs found in all the TCF family members. Each TCF activation domain can be phosphorylated in vitro by partially purified ERK2, and ERK activation in vivo is sufficient to potentiate transcriptional activation.

AF4 and AF4N protein complexes: recruitment of P-TEFb kinase, their interactome and potential functions

PubMed Central

Scholz, Bastian; Kowarz, Eric; Rössler, Tanja; Ahmad, Khalil; Steinhilber, Dieter; Marschalek, Rolf

2015-01-01

AF4/AFF1 and AF5/AFF4 are the molecular backbone to assemble “super-elongation complexes” (SECs) that have two main functions: (1) control of transcriptional elongation by recruiting the positive transcription elongation factor b (P-TEFb = CyclinT1/CDK9) that is usually stored in inhibitory 7SK RNPs; (2) binding of different histone methyltransferases, like DOT1L, NSD1 and CARM1. This way, transcribed genes obtain specific histone signatures (e.g. H3K79me2/3, H3K36me2) to generate a transcriptional memory system. Here we addressed several questions: how is P-TEFb recruited into SEC, how is the AF4 interactome composed, and what is the function of the naturally occuring AF4N protein variant which exhibits only the first 360 amino acids of the AF4 full-length protein. Noteworthy, shorter protein variants are a specific feature of all AFF protein family members. Here, we demonstrate that full-length AF4 and AF4N are both catalyzing the transition of P-TEFb from 7SK RNP to their N-terminal domain. We have also mapped the protein-protein interaction network within both complexes. In addition, we have first evidence that the AF4N protein also recruits TFIIH and the tumor suppressor MEN1. This indicate that AF4N may have additional functions in transcriptional initiation and in MEN1-dependend transcriptional processes. PMID:26171280

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1.

PubMed

Iizuka, Ryo; Sugano, Yuri; Ide, Naoki; Ohtaki, Akashi; Yoshida, Takao; Fujiwara, Shinsuke; Imanaka, Tadayuki; Yohda, Masafumi

2008-03-28

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two alpha subunits and four beta subunits with the structure of a double beta-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (alpha1, alpha2, beta1, and beta2) from T. KS-1. All of them (alpha1-beta1, alpha2-beta1, alpha1-beta2, and alpha2-beta2) exist as alpha(2)beta(4) heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the beta1 subunit interacted with the chaperonins more strongly than those with the beta2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.

Antibodies against multiple merozoite surface antigens of the human malaria parasite Plasmodium falciparum inhibit parasite maturation and red blood cell invasion.

PubMed

Woehlbier, Ute; Epp, Christian; Hackett, Fiona; Blackman, Michael J; Bujard, Hermann

2010-03-18

Plasmodium falciparum merozoites expose at their surface a large protein complex, which is composed of fragments of merozoite surface protein 1 (MSP-1; called MSP-183, MSP-130, MSP-138, and MSP-142) plus associated processing products of MSP-6 and MSP-7. During erythrocyte invasion this complex, as well as an integral membrane protein called apical membrane antigen-1 (AMA-1), is shed from the parasite surface following specific proteolysis. Components of the MSP-1/6/7 complex and AMA-1 are presently under development as malaria vaccines. The specificities and effects of antibodies directed against MSP-1, MSP-6, MSP-7 on the growth of blood stage parasites were studied using ELISA and the pLDH-assay. To understand the mode of action of these antibodies, their effects on processing of MSP-1 and AMA-1 on the surface of merozoites were investigated. Antibodies targeting epitopes located throughout the MSP-1/6/7 complex interfere with shedding of MSP-1, and as a consequence prevent erythrocyte invasion. Antibodies targeting the MSP-1/6/7 complex have no effect on the processing and shedding of AMA-1 and, similarly, antibodies blocking the shedding of AMA-1 do not affect cleavage of MSP-1, suggesting completely independent functions of these proteins during invasion. Furthermore, some epitopes, although eliciting highly inhibitory antibodies, are only poorly recognized by the immune system when presented in the structural context of the intact antigen. The findings reported provide further support for the development of vaccines based on MSP-1/6/7 and AMA-1, which would possibly include a combination of these antigens.

A wave-mechanical model of incoherent quasielastic scattering in complex systems.

PubMed

Frauenfelder, Hans; Fenimore, Paul W; Young, Robert D

2014-09-02

Quasielastic incoherent neutron scattering (QENS) is an important tool for the exploration of the dynamics of complex systems such as biomolecules, liquids, and glasses. The dynamics is reflected in the energy spectra of the scattered neutrons. Conventionally these spectra are decomposed into a narrow elastic line and a broad quasielastic band. The band is interpreted as being caused by Doppler broadening due to spatial motion of the target molecules. We propose a quantum-mechanical model in which there is no separate elastic line. The quasielastic band is composed of sharp lines with twice the natural line width, shifted from the center by a random walk of the protein in the free-energy landscape of the target molecule. The walk is driven by vibrations and by external fluctuations. We first explore the model with the Mössbauer effect. In the subsequent application to QENS we treat the incoming neutron as a de Broglie wave packet. While the wave packet passes the protons in the protein and the hydration shell it exchanges energy with the protein during the passage time of about 100 ns. The energy exchange broadens the ensemble spectrum. Because the exchange involves the free-energy landscape of the protein, the QENS not only provides insight into the protein dynamics, but it may also illuminate the free-energy landscape of the protein-solvent system.

The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

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

Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini

2012-03-26

Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC.more » While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.« less

Development of automated high throughput single molecular microfluidic detection platform for signal transduction analysis

NASA Astrophysics Data System (ADS)

Huang, Po-Jung; Baghbani Kordmahale, Sina; Chou, Chao-Kai; Yamaguchi, Hirohito; Hung, Mien-Chie; Kameoka, Jun

2016-03-01

Signal transductions including multiple protein post-translational modifications (PTM), protein-protein interactions (PPI), and protein-nucleic acid interaction (PNI) play critical roles for cell proliferation and differentiation that are directly related to the cancer biology. Traditional methods, like mass spectrometry, immunoprecipitation, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy require a large amount of sample and long processing time. "microchannel for multiple-parameter analysis of proteins in single-complex (mMAPS)"we proposed can reduce the process time and sample volume because this system is composed by microfluidic channels, fluorescence microscopy, and computerized data analysis. In this paper, we will present an automated mMAPS including integrated microfluidic device, automated stage and electrical relay for high-throughput clinical screening. Based on this result, we estimated that this automated detection system will be able to screen approximately 150 patient samples in a 24-hour period, providing a practical application to analyze tissue samples in a clinical setting.

Neurodegeneration in Alzheimer disease: role of amyloid precursor protein and presenilin 1 intracellular signaling.

PubMed

Nizzari, Mario; Thellung, Stefano; Corsaro, Alessandro; Villa, Valentina; Pagano, Aldo; Porcile, Carola; Russo, Claudio; Florio, Tullio

2012-01-01

Alzheimer disease (AD) is a heterogeneous neurodegenerative disorder characterized by (1) progressive loss of synapses and neurons, (2) intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3) amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2). The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration.

ATG13

PubMed Central

Alers, Sebastian; Wesselborg, Sebastian; Stork, Björn

2014-01-01

During the past 20 years, autophagy signaling has entered the main stage of the cell biological theater. Autophagy represents an intracellular degradation process that is involved in both the bulk recycling of cytoplasmic components and the selective removal of organelles, protein aggregates, or intracellular pathogens. The understanding of autophagy has been greatly facilitated by the characterization of the molecular machinery governing this process. In yeast, initiation of autophagy is controlled by the Atg1 kinase complex, which is composed of the Ser/Thr kinase Atg1, the adaptor protein Atg13, and the ternary complex of Atg17-Atg31-Atg29. In vertebrates, the orthologous ULK1 kinase complex contains the Ser/Thr kinase ULK1 and the accessory proteins ATG13, RB1CC1, and ATG101. Among these components, Atg1/ULK1 have gained major attention in the past, i.e., for the identification of upstream regulatory kinases, the characterization of downstream substrates controlling the autophagic flux, or as a druggable target for the modulation of autophagy. However, accumulating data indicate that the function of Atg13/ATG13 has been likely underestimated so far. In addition to ensuring proper Atg1/ULK1 recruitment and activity, this adaptor molecule has been implicated in ULK1-independent autophagy processes. Furthermore, recent data have identified additional binding partners of Atg13/ATG13 besides the components of the Atg1/ULK1 complex, e.g., Atg8 family proteins or acidic phospholipids. Therefore, in this review we will center the spotlight on Atg13/ATG13 and summarize the role that Atg13/ATG13 assumes in the autophagy stage play. PMID:24879146

Nuclear envelopathies: a complex LINC between nuclear envelope and pathology.

PubMed

Janin, Alexandre; Bauer, Delphine; Ratti, Francesca; Millat, Gilles; Méjat, Alexandre

2017-08-30

Since the identification of the first disease causing mutation in the gene coding for emerin, a transmembrane protein of the inner nuclear membrane, hundreds of mutations and variants have been found in genes encoding for nuclear envelope components. These proteins can be part of the inner nuclear membrane (INM), such as emerin or SUN proteins, outer nuclear membrane (ONM), such as Nesprins, or the nuclear lamina, such as lamins A and C. However, they physically interact with each other to insure the nuclear envelope integrity and mediate the interactions of the nuclear envelope with both the genome, on the inner side, and the cytoskeleton, on the outer side. The core of this complex, called LINC (LInker of Nucleoskeleton to Cytoskeleton) is composed of KASH and SUN homology domain proteins. SUN proteins are INM proteins which interact with lamins by their N-terminal domain and with the KASH domain of nesprins located in the ONM by their C-terminal domain.Although most of these proteins are ubiquitously expressed, their mutations have been associated with a large number of clinically unrelated pathologies affecting specific tissues. Moreover, variants in SUN proteins have been found to modulate the severity of diseases induced by mutations in other LINC components or interactors. For these reasons, the diagnosis and the identification of the molecular explanation of "nuclear envelopathies" is currently challenging.The aim of this review is to summarize the human diseases caused by mutations in genes coding for INM proteins, nuclear lamina, and ONM proteins, and to discuss their potential physiopathological mechanisms that could explain the large spectrum of observed symptoms.

Top-down venomics of the East African green mamba, Dendroaspis angusticeps, and the black mamba, Dendroaspis polylepis, highlight the complexity of their toxin arsenals.

PubMed

Petras, Daniel; Heiss, Paul; Harrison, Robert A; Süssmuth, Roderich D; Calvete, Juan J

2016-09-02

We report the characterization, by combination of high-resolution on-line molecular mass and disulfide bond profiling and top-down MS/MS analysis, of the venom proteomes of two congeneric African snake species of medical importance, Dendroaspis angusticeps (green mamba) and D. polylepis (black mamba). Each of these mamba venoms comprised more than two-hundred polypeptides belonging to just a few toxin families. Both venom proteomes are overwhelmingly composed of post-synaptically-acting short- and long-chain neurotoxins that potently inhibit muscle- and neuronal-type nicotinic acetylcholine receptors; muscarinic cardiotoxins; and dendrotoxins, that block some of the Kv1, n-class of K+ channels. However, the identity of the major proteins and their relative abundances exhibit marked interspecific variation. In addition, the greater resolution of the top-down venomic analytical approach revealed previously undetected protein species, isoforms and proteoforms, including the identification and precise location of modified lysine residues in a number of proteins in both venoms, but particularly in green mamba toxins. This comparative top-down venomic analysis unveiled the untapped complexity of Dendroaspis venoms and lays the foundations for rationalizing the notably different potency of green and black mamba lethal arsenals at locus resolution. We report the characterization, by combination of high-resolution on-line molecular mass and disulfide bond profiling and top-down MS/MS analysis, of the venom proteomes of two congeneric African snake species of medical importance, Dendroaspis angusticeps (green mamba) and D. polylepis (black mamba). Each of these mamba venoms comprised more than two-hundred polypeptides belonging to just a few toxin families. Both venom proteomes are overwhelmingly composed of post-synaptically-acting short- and long-chain neurotoxins that potently inhibit muscle- and neuronal-type nicotinic acetylcholine receptors; muscarinic cardiotoxins; and dendrotoxins, that block some of the Kv1, n-class of K+ channels. However, the identity of the major proteins and their relative abundances exhibit marked interspecific variation. In addition, the greater resolution of the top-down venomic analytical approach revealed previously undetected protein species, isoforms and proteoforms, including the identification and precise location of modified lysine residues in a number of proteins in both venoms, but particularly in green mamba toxins. This comparative top-down venomic analysis unveiled the untapped complexity of Dendroaspis venoms and lays the foundations for rationalizing the notably different potency of green and black mamba lethal arsenals at locus resolution.

Multiscale Modeling of Cardiac Cellular Energetics

PubMed Central

BASSINGTHWAIGHTE, JAMES B.; CHIZECK, HOWARD J.; ATLAS, LES E.; QIAN, HONG

2010-01-01

Multiscale modeling is essential to integrating knowledge of human physiology starting from genomics, molecular biology, and the environment through the levels of cells, tissues, and organs all the way to integrated systems behavior. The lowest levels concern biophysical and biochemical events. The higher levels of organization in tissues, organs, and organism are complex, representing the dynamically varying behavior of billions of cells interacting together. Models integrating cellular events into tissue and organ behavior are forced to resort to simplifications to minimize computational complexity, thus reducing the model’s ability to respond correctly to dynamic changes in external conditions. Adjustments at protein and gene regulatory levels shortchange the simplified higher-level representations. Our cell primitive is composed of a set of subcellular modules, each defining an intracellular function (action potential, tricarboxylic acid cycle, oxidative phosphorylation, glycolysis, calcium cycling, contraction, etc.), composing what we call the “eternal cell,” which assumes that there is neither proteolysis nor protein synthesis. Within the modules are elements describing each particular component (i.e., enzymatic reactions of assorted types, transporters, ionic channels, binding sites, etc.). Cell subregions are stirred tanks, linked by diffusional or transporter-mediated exchange. The modeling uses ordinary differential equations rather than stochastic or partial differential equations. This basic model is regarded as a primitive upon which to build models encompassing gene regulation, signaling, and long-term adaptations in structure and function. During simulation, simpler forms of the model are used, when possible, to reduce computation. However, when this results in error, the more complex and detailed modules and elements need to be employed to improve model realism. The processes of error recognition and of mapping between different levels of model form complexity are challenging but are essential for successful modeling of large-scale systems in reasonable time. Currently there is to this end no established methodology from computational sciences. PMID:16093514

Gene Composer: database software for protein construct design, codon engineering, and gene synthesis

PubMed Central

Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

2009-01-01

Background To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. Results An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. Conclusion We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease error correction in combination with PIPE cloning. In a sister manuscript we present data on how Gene Composer designed genes and protein constructs can result in improved protein production for structural studies. PMID:19383142

Gene composer: database software for protein construct design, codon engineering, and gene synthesis.

PubMed

Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

2009-04-21

To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease error correction in combination with PIPE cloning. In a sister manuscript we present data on how Gene Composer designed genes and protein constructs can result in improved protein production for structural studies.

Single Honeybee Silk Protein Mimics Properties of Multi-Protein Silk

PubMed Central

Sutherland, Tara D.; Church, Jeffrey S.; Hu, Xiao; Huson, Mickey G.; Kaplan, David L.; Weisman, Sarah

2011-01-01

Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk. PMID:21311767

Centromeric chromatin and its dynamics in plants.

PubMed

Lermontova, Inna; Sandmann, Michael; Mascher, Martin; Schmit, Anne-Catherine; Chabouté, Marie-Edith

2015-07-01

Centromeres are chromatin structures that are required for proper separation of chromosomes during mitosis and meiosis. The centromere is composed of centromeric DNA, often enriched in satellite repeats, and kinetochore complex proteins. To date, over 100 kinetochore components have been identified in various eukaryotes. Kinetochore assembly begins with incorporation of centromeric histone H3 variant CENH3 into centromeric nucleosomes. Protein components of the kinetochore are either present at centromeres throughout the cell cycle or localize to centromeres transiently, prior to attachment of microtubules to each kinetochore in prometaphase of mitotic cells. This is the case for the spindle assembly checkpoint (SAC) proteins in animal cells. The SAC complex ensures equal separation of chromosomes between daughter nuclei by preventing anaphase onset before metaphase is complete, i.e. the sister kinetochores of all chromosomes are attached to spindle fibers from opposite poles. In this review, we focus on the organization of centromeric DNA and the kinetochore assembly in plants. We summarize recent advances regarding loading of CENH3 into the centromere, and the subcellular localization and protein-protein interactions of Arabidopsis thaliana proteins involved in kinetochore assembly and function. We describe the transcriptional activity of corresponding genes based on in silico analysis of their promoters and cell cycle-dependent expression. Additionally, barley homologs of all selected A. thaliana proteins have been identified in silico, and their sequences and domain structures are presented. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

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

Timofeev, V. I., E-mail: inna@ns.crys.ras.ru, E-mail: tostars@mail.ru, E-mail: ugama@yandex.ru; Abramchik, Yu. A.; Zhukhlistova, N. E.

2016-01-15

Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sitesmore » were shown to be identical in both proteins.« less

Evidence of insulin-like growth factor binding protein-3 proteolysis during growth hormone stimulation testing.

PubMed

Nwosu, Benjamin U; Soyka, Leslie A; Angelescu, Amanda; Lee, Mary M

2011-01-01

The ternary complex is composed of insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3 and acid labile subunit (ALS). Growth hormone (GH) promotes IGFBP-3 proteolysis to release free IGF-I, ALS, and IGFBP-3 fragments. Our aim was to determine whether elevated GH levels during GH stimulation testing would trigger IGFBP-3 proteolysis. This prospective study of 10 short prepubertal children (height standard deviation score -2.37 +/- 0.31) used arginine and GH releasing hormone stimulation to study dynamic changes in the ternary complex moieties. IGFBP-3 was measured in two assays: a radioimmunoassay (RIA) that detects both cleaved and intact IGFBP-3; and an immunochemiluminescence assay (ICMA) that detects only intact IGFBP-3. IGFBP-3 measured by RIA increased by 19% (p < 0.05), while IGFBP-3 measured by ICMA did not significantly increase (6.1%). The significant increase in IGFBP-3 measured by RIA, but not ICMA, provides evidence of IGFBP-3 proteolysis during acute GH stimulation.

Prokaryotic cells: structural organisation of the cytoskeleton and organelles.

PubMed

Souza, Wanderley de

2012-05-01

For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

The human sirtuin family: Evolutionary divergences and functions

PubMed Central

2011-01-01

The sirtuin family of proteins is categorised as class III histone deacetylases that play complex and important roles in ageing-related pathological conditions such as cancer and the deregulation of metabolism. There are seven members in humans, divided into four classes, and evolutionarily conserved orthologues can be found in most forms of life, including both eukaryotes and prokaryotes. The highly conserved catalytic core domain composed of a large oxidised nicotinamide adenine dinucleotide (NAD+)-binding Rossmann fold subunit suggests that these proteins belong to a family of nutrient-sensing regulators. Along with their function in regulating cellular metabolism in response to stressful conditions, they are implicated in modifying a wide variety of substrates; this increases the complexity of unravelling the interplay of sirtuins and their partners. Over the past few years, all of these new findings have attracted the interest of researchers exploring potential therapeutic implications related to the function of sirtuins. It remains to be elucidated whether, indeed, sirtuins can serve as molecular targets for the treatment of human illnesses. PMID:21807603

A major protein component of the Bacillus subtilis biofilm matrix.

PubMed

Branda, Steven S; Chu, Frances; Kearns, Daniel B; Losick, Richard; Kolter, Roberto

2006-02-01

Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

Decoding Mechanisms by which Silent Codon Changes Influence Protein Biogenesis and Function

PubMed Central

Bali, Vedrana; Bebok, Zsuzsanna

2015-01-01

Scope Synonymous codon usage has been a focus of investigation since the discovery of the genetic code and its redundancy. The occurrences of synonymous codons vary between species and within genes of the same genome, known as codon usage bias. Today, bioinformatics and experimental data allow us to compose a global view of the mechanisms by which the redundancy of the genetic code contributes to the complexity of biological systems from affecting survival in prokaryotes, to fine tuning the structure and function of proteins in higher eukaryotes. Studies analyzing the consequences of synonymous codon changes in different organisms have revealed that they impact nucleic acid stability, protein levels, structure and function without altering amino acid sequence. As such, synonymous mutations inevitably contribute to the pathogenesis of complex human diseases. Yet, fundamental questions remain unresolved regarding the impact of silent mutations in human disorders. In the present review we describe developments in this area concentrating on mechanisms by which synonymous mutations may affect protein function and human health. Purpose This synopsis illustrates the significance of synonymous mutations in disease pathogenesis. We review the different steps of gene expression affected by silent mutations, and assess the benefits and possible harmful effects of codon optimization applied in the development of therapeutic biologics. Physiological and medical relevance Understanding mechanisms by which synonymous mutations contribute to complex diseases such as cancer, neurodegeneration and genetic disorders, including the limitations of codon-optimized biologics, provides insight concerning interpretation of silent variants and future molecular therapies. PMID:25817479

Regulated transport into the nucleus of herpesviridae DNA replication core proteins.

PubMed

Gualtiero, Alvisi; Jans, David A; Camozzi, Daria; Avanzi, Simone; Loregian, Arianna; Ripalti, Alessandro; Palù, Giorgio

2013-09-16

The Herpesvirdae family comprises several major human pathogens belonging to three distinct subfamilies. Their double stranded DNA genome is replicated in the nuclei of infected cells by a number of host and viral products. Among the latter the viral replication complex, whose activity is strictly required for viral replication, is composed of six different polypeptides, including a two-subunit DNA polymerase holoenzyme, a trimeric primase/helicase complex and a single stranded DNA binding protein. The study of herpesviral DNA replication machinery is extremely important, both because it provides an excellent model to understand processes related to eukaryotic DNA replication and it has important implications for the development of highly needed antiviral agents. Even though all known herpesviruses utilize very similar mechanisms for amplification of their genomes, the nuclear import of the replication complex components appears to be a heterogeneous and highly regulated process to ensure the correct spatiotemporal localization of each protein. The nuclear transport process of these enzymes is controlled by three mechanisms, typifying the main processes through which protein nuclear import is generally regulated in eukaryotic cells. These include cargo post-translational modification-based recognition by the intracellular transporters, piggy-back events allowing coordinated nuclear import of multimeric holoenzymes, and chaperone-assisted nuclear import of specific subunits. In this review we summarize these mechanisms and discuss potential implications for the development of antiviral compounds aimed at inhibiting the Herpesvirus life cycle by targeting nuclear import of the Herpesvirus DNA replicating enzymes.

Crystal structure of LysK, an enzyme catalyzing the last step of lysine biosynthesis in Thermus thermophilus, in complex with lysine: Insight into the mechanism for recognition of the amino-group carrier protein, LysW.

PubMed

Fujita, Satomi; Cho, Su-Hee; Yoshida, Ayako; Hasebe, Fumihito; Tomita, Takeo; Kuzuyama, Tomohisa; Nishiyama, Makoto

2017-09-16

LysK is an M20 peptidase family enzyme that hydrolyzes the isopeptide bond between the carrier protein LysW and lysine in order to release lysine, which is the last step of lysine biosynthesis in Thermus thermophilus. In the present study, we determined the crystal structure of LysK in complex with lysine at a resolution of 2.4 Å. The α-amino group of the bound lysine was oriented toward the catalytic center, which was composed of the residues coordinating divalent metal ions for the hydrolysis of the isopeptide bond. An 11 Å-long path was observed from the active site binding lysine to the protein surface, which may be responsible for recognizing the C-terminal extension domain of LysW with the conserved EDWGE sequence. A positively-charged surface region was detected around the exit of the path, similar to other lysine biosynthetic enzymes using LysW as the carrier protein. Mutational studies of the surface residues provided a plausible model for the electrostatic interaction with LysW. Copyright © 2017 Elsevier Inc. All rights reserved.

Hsp90α forms a stable complex at the cilium neck for the interaction of signalling molecules in IGF-1 receptor signalling.

PubMed

Wang, Hongzhong; Zou, Xinle; Wei, Zhuang; Wu, Yuan; Li, Rongxia; Zeng, Rong; Chen, Zhengjun; Liao, Kan

2015-01-01

The primary cilium is composed of an axoneme that protrudes from the cell surface, a basal body beneath the membrane and a transition neck in between. It is a sensory organelle on the plasma membrane, involved in mediating extracellular signals. In the transition neck region of the cilium, the microtubules change from triplet to doublet microtubules. This region also contains the transition fibres that crosslink the axoneme with the membrane and the necklace proteins that regulate molecules being transported into and out of the cilium. In this protein-enriched, complex area it is important to maintain the correct assembly of all of these proteins. Here, through immunofluorescent staining and protein isolation, we identify the molecular chaperone Hsp90α clustered at the periciliary base. At the transition neck region, phosphorylated Hsp90α forms a stable ring around the axoneme. Heat shock treatment causes Hsp90α to dissipate and induces resorption of cilia. We further identify that Hsp90α at the transition neck region represents a signalling platform on which IRS-1 interacts with intracellular downstream signalling molecules involved in IGF-1 receptor signalling. © 2015. Published by The Company of Biologists Ltd.

Characterization of the Grp94/OS-9 chaperone-lectin complex

PubMed Central

Seidler, Paul M.; Shinsky, Stephen A.; Hong, Feng; Li, Zihai; Cosgrove, Michael S.; Gewirth, Daniel T.

2014-01-01

Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER associated degradation (ERAD) quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins (TMPs). To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal MRH lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains ‘mammalian-specific insets’ that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate, and additionally hints at a plausible biological role for the Grp94/OS-9 complex. PMID:25193139

Insight into the composition of the intercellular matrix of Streptococcus pneumoniae biofilms.

PubMed

Domenech, Mirian; García, Ernesto; Prieto, Alicia; Moscoso, Miriam

2013-02-01

Biofilm matrices consist of a mixture of extracellular polymeric substances synthesized in large part by the biofilm-producing microorganisms themselves. These matrices are responsible for the cohesion and three-dimensional architecture of biofilms. The present study demonstrates the existence of a matrix composed of extracellular DNA, proteins and polysaccharides in the biofilm formed by the human pathogen Streptococcus pneumoniae. Extracellular DNA, visualized by fluorescent labelling, was an important component of this matrix. The existence of DNA-protein complexes associated with bacterial aggregates and other polymers was hypothesized based on the unexpected DNA binding activity of lysozyme LytC, a novel moonlighting protein. Actually, a 25-amino-acid-long peptide derived from LytC (positions 408 and 432 of the mature LytC) was also capable of efficiently binding to DNA. Moreover, the presence of intercellular DNA-LytC protein complexes in pneumococcal biofilms was demonstrated by confocal laser scanning microscopy. Evidence of extracellular polysaccharide different from the capsule was obtained by staining with Calcofluor dye and four types of lectin conjugated to Alexa fluorophores, and by incubation with glycoside hydrolases. The presence of residues of Glcp(1→4) and GlcNAc(1→4) (in its deacetylated form) in the pneumococcal biofilm was confirmed by GC-MS techniques. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Ram seminal plasma proteome and its impact on liquid preservation of spermatozoa.

PubMed

Soleilhavoup, C; Tsikis, G; Labas, V; Harichaux, G; Kohnke, P L; Dacheux, J L; Guérin, Y; Gatti, J L; de Graaf, S P; Druart, X

2014-09-23

Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC-MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24h in milk at 15°C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24h preservation period. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA-mediated engineering of multicomponent enzyme crystals

PubMed Central

Brodin, Jeffrey D.; Auyeung, Evelyn; Mirkin, Chad A.

2015-01-01

The ability to predictably control the coassembly of multiple nanoscale building blocks, especially those with disparate chemical and physical properties such as biomolecules and inorganic nanoparticles, has far-reaching implications in catalysis, sensing, and photonics, but a generalizable strategy for engineering specific contacts between these particles is an outstanding challenge. This is especially true in the case of proteins, where the types of possible interparticle interactions are numerous, diverse, and complex. Herein, we explore the concept of trading protein–protein interactions for DNA–DNA interactions to direct the assembly of two nucleic-acid–functionalized proteins with distinct surface chemistries into six unique lattices composed of catalytically active proteins, or of a combination of proteins and DNA-modified gold nanoparticles. The programmable nature of DNA–DNA interactions used in this strategy allows us to control the lattice symmetries and unit cell constants, as well as the compositions and habit, of the resulting crystals. This study provides a potentially generalizable strategy for constructing a unique class of materials that take advantage of the diverse morphologies, surface chemistries, and functionalities of proteins for assembling functional crystalline materials. PMID:25831510

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation

PubMed Central

Kugeratski, Fernanda G; Batista, Michel; Inoue, Alexandre Haruo; Ramos, Bruno Dias; Krieger, Marco Aurelio; Marchini/, Fabricio K

2015-01-01

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzi genes and is freely available for scientific community. PMID:26200713

DNA-mediated engineering of multicomponent enzyme crystals

DOE PAGES

Brodin, Jeffrey D.; Auyeung, Evelyn; Mirkin, Chad A.

2015-03-23

The ability to predictably control the coassembly of multiple nanoscale building blocks, especially those with disparate chemical and physical properties such as biomolecules and inorganic nanoparticles, has far-reaching implications in catalysis, sensing, and photonics, but a generalizable strategy for engineering specific contacts between these particles is an outstanding challenge. This is especially true in the case of proteins, where the types of possible interparticle interactions are numerous, diverse, and complex. In this paper, we explore the concept of trading protein–protein interactions for DNA–DNA interactions to direct the assembly of two nucleic-acid–functionalized proteins with distinct surface chemistries into six unique latticesmore » composed of catalytically active proteins, or of a combination of proteins and DNA-modified gold nanoparticles. The programmable nature of DNA–DNA interactions used in this strategy allows us to control the lattice symmetries and unit cell constants, as well as the compositions and habit, of the resulting crystals. Finally, this study provides a potentially generalizable strategy for constructing a unique class of materials that take advantage of the diverse morphologies, surface chemistries, and functionalities of proteins for assembling functional crystalline materials.« less

Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System.

PubMed

Schwarz-Schilling, Matthaeus; Dupin, Aurore; Chizzolini, Fabio; Krishnan, Swati; Mansy, Sheref S; Simmel, Friedrich C

2018-04-11

Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.

A quasi-atomic model of human adenovirus type 5 capsid

PubMed Central

Fabry, Céline M S; Rosa-Calatrava, Manuel; Conway, James F; Zubieta, Chloé; Cusack, Stephen; Ruigrok, Rob W H; Schoehn, Guy

2005-01-01

Adenoviruses infect a wide range of vertebrates including humans. Their icosahedral capsids are composed of three major proteins: the trimeric hexon forms the facets and the penton, a noncovalent complex of the pentameric penton base and trimeric fibre proteins, is located at the 12 capsid vertices. Several proteins (IIIa, VI, VIII and IX) stabilise the capsid. We have obtained a 10 Å resolution map of the human adenovirus 5 by image analysis from cryo-electron micrographs (cryoEMs). This map, in combination with the X-ray structures of the penton base and hexon, was used to build a quasi-atomic model of the arrangement of the two major capsid components and to analyse the hexon–hexon and hexon–penton interactions. The secondary proteins, notably VIII, were located by comparing cryoEM maps of native and pIX deletion mutant virions. Minor proteins IX and IIIa are located on the outside of the capsid, whereas protein VIII is organised with a T=2 lattice on the inner face of the capsid. The capsid organisation is compared with the known X-ray structure of bacteriophage PRD1. PMID:15861131

Ethanol and Protein from Ethanol Plant By-Products Using Edible Fungi Neurospora intermedia and Aspergillus oryzae.

PubMed

Bátori, Veronika; Ferreira, Jorge A; Taherzadeh, Mohammad J; Lennartsson, Patrik R

2015-01-01

Feasible biorefineries for production of second-generation ethanol are difficult to establish due to the process complexity. An alternative is to partially include the process in the first-generation plants. Whole stillage, a by-product from dry-mill ethanol processes from grains, is mostly composed of undegraded bran and lignocelluloses can be used as a potential substrate for production of ethanol and feed proteins. Ethanol production and the proteins from the stillage were investigated using the edible fungi Neurospora intermedia and Aspergillus oryzae, respectively. N. intermedia produced 4.7 g/L ethanol from the stillage and increased to 8.7 g/L by adding 1 FPU of cellulase/g suspended solids. Saccharomyces cerevisiae produced 0.4 and 5.1 g/L ethanol, respectively. Under a two-stage cultivation with both fungi, up to 7.6 g/L of ethanol and 5.8 g/L of biomass containing 42% (w/w) crude protein were obtained. Both fungi degraded complex substrates including arabinan, glucan, mannan, and xylan where reductions of 91, 73, 38, and 89% (w/v) were achieved, respectively. The inclusion of the current process can lead to the production of 44,000 m(3) of ethanol (22% improvement), around 12,000 tons of protein-rich biomass for animal feed, and energy savings considering a typical facility producing 200,000 m(3) ethanol/year.

Identification of dynamin as a septin-binding protein.

PubMed

Maimaitiyiming, Maowulan; Kobayashi, Yuumi; Kumanogoh, Haruko; Nakamura, Shun; Morita, Mitsuhiro; Maekawa, Shohei

2013-02-08

Lipid rafts (detergent-resistant low-density membrane microdomain: DRM) are signal-transducing membrane platforms. In a previous study, we showed maturation-dependent localization of septin in the DRM fraction of rat brain. Mammalian septin is composed with 13-14 isoforms and these isoforms assemble to form rod-shaped hetero-oligomeric complexes. End-to-end polymerization of these complexes results in the formation of higher order structures such as filamentous sheets or bundles of filaments that restrict the fluid-like diffusion of the membrane proteins and lipids. Considering the function of septin as the membrane scaffold, elucidation of the molecular interaction of septin in DRM could be a breakthrough to understand another role of lipid rafts. In order to identify septin-binding proteins in DRM, solubilization and fractionation of septin from DRM was attempted. Several proteins were co-fractionated with septin and LC-MS/MS analysis identified one of these proteins as dynamin and Western blotting using anti-dynamin confirmed this result. Immunoprecipitation of septin11 in a crude supernatant showed co-precipitation of dynamin and dynamin fraction prepared from brain contained several septin isoforms. Within bacterially expressed septin isoforms, septin5 and septin11 bound dynamin but septin9 did not. These results suggest that some septin isoforms participate in the dynamin-related membrane dynamics. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Altered Protein Interactions of the Endogenous Interactome of PTPIP51 towards MAPK Signaling

PubMed Central

Brobeil, Alexander; Chehab, Rajaa; Dietel, Eric; Gattenlöhner, Stefan; Wimmer, Monika

2017-01-01

Protein–protein interactions play a pivotal role in normal cellular functions as well as in carcinogenesis. The protein–protein interactions form functional clusters during signal transduction. To elucidate the fine calibration of the protein–protein interactions of protein tyrosine phosphatase interacting protein 51 (PTPIP51) a small molecule drug, namely LDC-3, directly targeting PTPIP51 is now available. Therefore, LDC-3 allows for the studying of the regulation of the endogenous interactome by modulating PTPIP51 binding capacity. Small interfering ribonucleic acid (siRNA) experiments show that the modification in PTPIP51 binding capacity is induced by LDC-3. Application of LDC-3 annuls the known regulatory phosphorylation mechanisms for PTPIP51 and consequently, significantly alters the assembly of the PTPIP51 associated protein complexes. The treatment of human keratinocytes (HaCaT cells) with LDC-3 induces an altered protein–protein interaction profile of the endogenous interactome of PTPIP51. In addition, LDC-3 stabilizes PTPIP51 within a mitogen activated protein kinase (MAPK) complex composed of Raf-1 and the scaffold protein 14-3-3, independent of the phosphorylation status of PTPIP51. Of note, under LDC-3 treatment the regulatory function of the PTP1B on PTPIP51 fails to impact the PTPIP51 interaction characteristics, as reported for the HaCaT cell line. In summary, LDC-3 gives the unique opportunity to directly modulate PTPIP51 in malignant cells, thus targeting potential dysregulated signal transduction pathways such as the MAPK cascade. The provided data give critical insights in the therapeutic potential of PTPIP51 protein interactions and thus are basic for possible targeted therapy regimens. PMID:28754031

Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex [corrected].

PubMed

Palumbo, Valeria; Pellacani, Claudia; Heesom, Kate J; Rogala, Kacper B; Deane, Charlotte M; Mottier-Pavie, Violaine; Gatti, Maurizio; Bonaccorsi, Silvia; Wakefield, James G

2015-06-29

Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Clostridium and bacillus binary enterotoxins: bad for the bowels, and eukaryotic being.

PubMed

Stiles, Bradley G; Pradhan, Kisha; Fleming, Jodie M; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R

2014-09-05

Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin.

Clostridium and Bacillus Binary Enterotoxins: Bad for the Bowels, and Eukaryotic Being

PubMed Central

Stiles, Bradley G.; Pradhan, Kisha; Fleming, Jodie M.; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R.

2014-01-01

Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin. PMID:25198129

Characterization of the archaeal ribonuclease P proteins from Pyrococcus horikoshii OT3.

PubMed

Terada, Atsushi; Honda, Takashi; Fukuhara, Hideo; Hada, Kazumasa; Kimura, Makoto

2006-08-01

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5'-leader sequence of precursor tRNA (pre-tRNA). Our earlier study revealed that RNase P RNA (pRNA) and five proteins (PhoPop5, PhoRpp38, PhoRpp21, PhoRpp29, and PhoRpp30) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 reconstituted RNase P activity that exhibits enzymatic properties like those of the authentic enzyme. In present study, we investigated involvement of the individual proteins in RNase P activity. Two particles (R-3Ps), in which pRNA was mixed with three proteins, PhoPop5, PhoRpp30, and PhoRpp38 or PhoPop5, PhoRpp30, and PhoRpp21 showed a detectable RNase P activity, and five reconstituted particles (R-4Ps) composed of pRNA and four proteins exhibited RNase P activity, albeit at reduced level compared to that of the reconstituted particle (R-5P) composed of pRNA and five proteins. Time-course analysis of the RNase P activities of R-4Ps indicated that the R-4Ps lacking PhoPop5, PhoRpp21, or PhoRpp30 had virtually reduced activity, while omission of PhoRpp29 or PhoRpp38 had a slight effect on the activity. The results indicate that the proteins contribute to RNase P activity in order of PhoPop5 > PhoRpp30 > PhoRpp21 > PhoRpp29 > PhoRpp38. It was further found that R-4Ps showed a characteristic Mg2+ ion dependency approximately identical to that of R-5P. However, R-4Ps had optimum temperature of around at 55 degrees C which is lower than 70 degrees C for R-5P. Together, it is suggested that the P. horikoshii RNase P proteins are predominantly involved in optimization of the pRNA conformation, though they are individually dispensable for RNase P activity in vitro.

Global Motions of the Nuclear Pore Complex: Insights from Elastic Network Models

PubMed Central

Lezon, Timothy R.; Sali, Andrej; Bahar, Ivet

2009-01-01

The nuclear pore complex (NPC) is the gate to the nucleus. Recent determination of the configuration of proteins in the yeast NPC at ∼5 nm resolution permits us to study the NPC global dynamics using coarse-grained structural models. We investigate these large-scale motions by using an extended elastic network model (ENM) formalism applied to several coarse-grained representations of the NPC. Two types of collective motions (global modes) are predicted by the ENMs to be intrinsically favored by the NPC architecture: global bending and extension/contraction from circular to elliptical shapes. These motions are shown to be robust against tested variations in the representation of the NPC, and are largely captured by a simple model of a toroid with axially varying mass density. We demonstrate that spoke multiplicity significantly affects the accessible number of symmetric low-energy modes of motion; the NPC-like toroidal structures composed of 8 spokes have access to highly cooperative symmetric motions that are inaccessible to toroids composed of 7 or 9 spokes. The analysis reveals modes of motion that may facilitate macromolecular transport through the NPC, consistent with previous experimental observations. PMID:19730674

Global motions of the nuclear pore complex: insights from elastic network models.

PubMed

Lezon, Timothy R; Sali, Andrej; Bahar, Ivet

2009-09-01

The nuclear pore complex (NPC) is the gate to the nucleus. Recent determination of the configuration of proteins in the yeast NPC at approximately 5 nm resolution permits us to study the NPC global dynamics using coarse-grained structural models. We investigate these large-scale motions by using an extended elastic network model (ENM) formalism applied to several coarse-grained representations of the NPC. Two types of collective motions (global modes) are predicted by the ENMs to be intrinsically favored by the NPC architecture: global bending and extension/contraction from circular to elliptical shapes. These motions are shown to be robust against tested variations in the representation of the NPC, and are largely captured by a simple model of a toroid with axially varying mass density. We demonstrate that spoke multiplicity significantly affects the accessible number of symmetric low-energy modes of motion; the NPC-like toroidal structures composed of 8 spokes have access to highly cooperative symmetric motions that are inaccessible to toroids composed of 7 or 9 spokes. The analysis reveals modes of motion that may facilitate macromolecular transport through the NPC, consistent with previous experimental observations.

Genetic analysis of the contribution of LTBP-3 to thoracic aneurysm in Marfan syndrome

PubMed Central

Zilberberg, Lior; Phoon, Colin K. L.; Robertson, Ian; Dabovic, Branka; Ramirez, Francesco; Rifkin, Daniel B.

2015-01-01

Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue, caused by mutations of the microfibrillar protein fibrillin-1, that predisposes affected individuals to aortic aneurysm and rupture and is associated with increased TGFβ signaling. TGFβ is secreted from cells as a latent complex consisting of TGFβ, the TGFβ propeptide, and a molecule of latent TGFβ binding protein (LTBP). Improper extracellular localization of the latent complex can alter active TGFβ levels, and has been hypothesized as an explanation for enhanced TGFβ signaling observed in MFS. We previously reported the absence of LTBP-3 in matrices lacking fibrillin-1, suggesting that perturbed TGFβ signaling in MFS might be due to defective interaction of latent TGFβ complexes containing LTBP-3 with mutant fibrillin-1 microfibrils. To test this hypothesis, we genetically suppressed Ltbp3 expression in a mouse model of progressively severe MFS. Here, we present evidence that MFS mice lacking LTBP-3 have improved survival, essentially no aneurysms, reduced disruption and fragmentation of medial elastic fibers, and decreased Smad2/3 and Erk1/2 activation in their aortas. These data suggest that, in MFS, improper localization of latent TGFβ complexes composed of LTBP-3 and TGFβ contributes to aortic disease progression. PMID:26494287

Chromatin signaling to kinetochores: Trans-regulation of Dam1 methylation by histone H2B ubiquitination

PubMed Central

Latham, John A.; Chosed, Renée J.; Wang, Shanzhi; Dent, Sharon Y.R.

2011-01-01

Summary Histone H3K4 trimethylation by the Set1/MLL family of proteins provides a hallmark for transcriptional activity from yeast to humans. In S. cerevisiae, H3K4 methylation is mediated by the Set1-containing COMPASS complex and is regulated in trans by prior ubiquitination of histone H2BK123. All of the events that regulate H2BK123ub and H3K4me are thought to occur at gene promoters. Here we report that this pathway is indispensable for methylation of the only other known substrate of Set1, K233 in Dam1, at kinetochores. Deletion of RAD6, BRE1, or Paf1 complex members abolishes Dam1 methylation, as does mutation of H2BK123. Our results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription. Moreover, our data identify a node of regulatory cross-talk in trans between a histone modification and modification on a non-histone protein, demonstrating that changing chromatin states can signal functional changes in other essential cellular proteins and machineries. PMID:21884933

Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

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

Sampathkumar, Parthasarathy; Kim, Seung Joong; Manglicmot, Danalyn

2012-10-23

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of {approx}456 polypeptide chains contributed by {approx}30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal 'FG' repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain ofmore » Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 {angstrom} resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.« less

Synaptogenesis Is Modulated by Heparan Sulfate in Caenorhabditis elegans

PubMed Central

Lázaro-Peña, María I.; Díaz-Balzac, Carlos A.; Bülow, Hannes E.; Emmons, Scott W.

2018-01-01

The nervous system regulates complex behaviors through a network of neurons interconnected by synapses. How specific synaptic connections are genetically determined is still unclear. Male mating is the most complex behavior in Caenorhabditis elegans. It is composed of sequential steps that are governed by > 3000 chemical connections. Here, we show that heparan sulfates (HS) play a role in the formation and function of the male neural network. HS, sulfated in position 3 by the HS modification enzyme HST-3.1/HS 3-O-sulfotransferase and attached to the HS proteoglycan glypicans LON-2/glypican and GPN-1/glypican, functions cell-autonomously and nonautonomously for response to hermaphrodite contact during mating. Loss of 3-O sulfation resulted in the presynaptic accumulation of RAB-3, a molecule that localizes to synaptic vesicles, and disrupted the formation of synapses in a component of the mating circuits. We also show that the neural cell adhesion protein NRX-1/neurexin promotes and the neural cell adhesion protein NLG-1/neuroligin inhibits the formation of the same set of synapses in a parallel pathway. Thus, neural cell adhesion proteins and extracellular matrix components act together in the formation of synaptic connections. PMID:29559501

Computational multiscale modeling in protein--ligand docking.

PubMed

Taufer, Michela; Armen, Roger; Chen, Jianhan; Teller, Patricia; Brooks, Charles

2009-01-01

In biological systems, the binding of small molecule ligands to proteins is a crucial process for almost every aspect of biochemistry and molecular biology. Enzymes are proteins that function by catalyzing specific biochemical reactions that convert reactants into products. Complex organisms are typically composed of cells in which thousands of enzymes participate in complex and interconnected biochemical pathways. Some enzymes serve as sequential steps in specific pathways (such as energy metabolism), while others function to regulate entire pathways and cellular functions [1]. Small molecule ligands can be designed to bind to a specific enzyme and inhibit the biochemical reaction. Inhibiting the activity of key enzymes may result in the entire biochemical pathways being turned on or off [2], [3]. Many small molecule drugs marketed today function in this generic way as enzyme inhibitors. If research identifies a specific enzyme as being crucial to the progress of disease, then this enzyme may be targeted with an inhibitor, which may slow down or reverse the progress of disease. In this way, enzymes are targeted from specific pathogens (e.g., virus, bacteria, fungi) for infectious diseases [4], [5], and human enzymes are targeted for noninfectious diseases such as cardiovascular disease, cancer, diabetes, and neurodegenerative diseases [6].

Hybrid protein-synthetic polymer nanoparticles for drug delivery.

PubMed

Koseva, Neli S; Rydz, Joanna; Stoyanova, Ekaterina V; Mitova, Violeta A

2015-01-01

Among the most common nanoparticulate systems, the polymeric nanocarriers have a number of key benefits, which give a great choice of delivery platforms. Nevertheless, polymeric nanoparticles possess some limitations that include use of toxic solvents in the production process, polymer degradation, drug leakage outside the diseased tissue, and polymer cytotoxicity. The combination of polymers of biological and synthetic origin is an appealing modern strategy for the production of novel nanocarriers with unprecedented properties. Proteins' interface can play an important role in determining bioactivity and toxicity and gives perspective for future development of the polymer-based nanoparticles. The design of hybrid constructs composed of synthetic polymer and biological molecules such as proteins can be considered as a straightforward tool to integrate a broad spectrum of properties and biofunctions into a single device. This review discusses hybrid protein-synthetic polymer nanoparticles with different structures and levels in complexity and functionality, in view of their applications as drug delivery systems. © 2015 Elsevier Inc. All rights reserved.

Bcs1p can rescue a large and productive cytochrome bc(1) complex assembly intermediate in the inner membrane of yeast mitochondria.

PubMed

Conte, Laura; Trumpower, Bernard L; Zara, Vincenzo

2011-01-01

The yeast cytochrome bc(1) complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc(1) complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc(1) assembly and the formation of a functionally inactive bc(1) core structure of about 500-kDa. This immature bc(1) core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc(1) core structure leading to the formation of the functional homodimeric bc(1) complex. Following Bcs1p expression, the mature bc(1) complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc(1) complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc(1) complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc(1) core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc(1) complex and gives new insights into the molecular mechanisms involved in the last steps of bc(1) assembly. Copyright © 2010 Elsevier B.V. All rights reserved.

Loss of the mitochondrial protein-only ribonuclease P complex causes aberrant tRNA processing and lethality in Drosophila.

PubMed

Sen, Aditya; Karasik, Agnes; Shanmuganathan, Aranganathan; Mirkovic, Elena; Koutmos, Markos; Cox, Rachel T

2016-07-27

Proteins encoded by mitochondrial DNA are translated using mitochondrially encoded tRNAs and rRNAs. As with nuclear encoded tRNAs, mitochondrial tRNAs must be processed to become fully functional. The mitochondrial form of ribonuclease P (mt:RNase P) is responsible for 5'-end maturation and is comprised of three proteins; mitochondrial RNase P protein (MRPP) 1 and 2 together with proteinaceous RNase P (PRORP). However, its mechanism and impact on development is not yet known. Using homology searches, we have identified the three proteins composing Drosophila mt:RNase P: Mulder (PRORP), Scully (MRPP2) and Roswell (MRPP1). Here, we show that each protein is essential and localizes with mitochondria. Furthermore, reducing levels of each causes mitochondrial deficits, which appear to be due at least in part to defective mitochondrial tRNA processing. Overexpressing two members of the complex, Mulder and Roswell, is also lethal, and in the case of Mulder, causes abnormal mitochondrial morphology. These data are the first evidence that defective mt:RNase P causes mitochondrial dysfunction, lethality and aberrant mitochondrial tRNA processing in vivo, underscoring its physiological importance. This in vivo mt:RNase P model will advance our understanding of how loss of mitochondrial tRNA processing causes tissue failure, an important aspect of human mitochondrial disease. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I

PubMed Central

1995-01-01

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein. PMID:7860628

Direct interaction of the bacteriophage SPP1 packaging ATPase with the portal protein.

PubMed

Oliveira, Leonor; Cuervo, Ana; Tavares, Paulo

2010-03-05

DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.

ATG13: just a companion, or an executor of the autophagic program?

PubMed

Alers, Sebastian; Wesselborg, Sebastian; Stork, Björn

2014-06-01

During the past 20 years, autophagy signaling has entered the main stage of the cell biological theater. Autophagy represents an intracellular degradation process that is involved in both the bulk recycling of cytoplasmic components and the selective removal of organelles, protein aggregates, or intracellular pathogens. The understanding of autophagy has been greatly facilitated by the characterization of the molecular machinery governing this process. In yeast, initiation of autophagy is controlled by the Atg1 kinase complex, which is composed of the Ser/Thr kinase Atg1, the adaptor protein Atg13, and the ternary complex of Atg17-Atg31-Atg29. In vertebrates, the orthologous ULK1 kinase complex contains the Ser/Thr kinase ULK1 and the accessory proteins ATG13, RB1CC1, and ATG101. Among these components, Atg1/ULK1 have gained major attention in the past, i.e., for the identification of upstream regulatory kinases, the characterization of downstream substrates controlling the autophagic flux, or as a druggable target for the modulation of autophagy. However, accumulating data indicate that the function of Atg13/ATG13 has been likely underestimated so far. In addition to ensuring proper Atg1/ULK1 recruitment and activity, this adaptor molecule has been implicated in ULK1-independent autophagy processes. Furthermore, recent data have identified additional binding partners of Atg13/ATG13 besides the components of the Atg1/ULK1 complex, e.g., Atg8 family proteins or acidic phospholipids. Therefore, in this review we will center the spotlight on Atg13/ATG13 and summarize the role that Atg13/ATG13 assumes in the autophagy stage play.

Hemoglobin–Albumin Cluster Incorporating a Pt Nanoparticle: Artificial O2 Carrier with Antioxidant Activities

PubMed Central

Hosaka, Hitomi; Haruki, Risa; Yamada, Kana; Böttcher, Christoph; Komatsu, Teruyuki

2014-01-01

A covalent core–shell structured protein cluster composed of hemoglobin (Hb) at the center and human serum albumins (HSA) at the periphery, Hb-HSAm, is an artificial O2 carrier that can function as a red blood cell substitute. Here we described the preparation of a novel Hb-HSA3 cluster with antioxidant activities and its O2 complex stable in aqueous H2O2 solution. We used an approach of incorporating a Pt nanoparticle (PtNP) into the exterior HSA unit of the cluster. A citrate reduced PtNP (1.8 nm diameter) was bound tightly within the cleft of free HSA with a binding constant (K) of 1.1×107 M−1, generating a stable HSA-PtNP complex. This platinated protein showed high catalytic activities for dismutations of superoxide radical anions (O2 •–) and hydrogen peroxide (H2O2), i.e., superoxide dismutase and catalase activities. Also, Hb-HSA3 captured PtNP into the external albumin unit (K = 1.1×107 M−1), yielding an Hb-HSA3(PtNP) cluster. The association of PtNP caused no alteration of the protein surface net charge and O2 binding affinity. The peripheral HSA-PtNP shell prevents oxidation of the core Hb, which enables the formation of an extremely stable O2 complex, even in H2O2 solution. PMID:25310133

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

PubMed Central

Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

2014-01-01

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

PubMed

Hiraga, Shin-Ichiro; Alvino, Gina M; Chang, Fujung; Lian, Hui-Yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J; Weinreich, Michael; Raghuraman, M K; Donaldson, Anne D

2014-02-15

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

Germline-specific H1 variants: the "sexy" linker histones.

PubMed

Pérez-Montero, Salvador; Carbonell, Albert; Azorín, Fernando

2016-03-01

The eukaryotic genome is packed into chromatin, a nucleoprotein complex mainly formed by the interaction of DNA with the abundant basic histone proteins. The fundamental structural and functional subunit of chromatin is the nucleosome core particle, which is composed by 146 bp of DNA wrapped around an octameric protein complex formed by two copies of each core histone H2A, H2B, H3, and H4. In addition, although not an intrinsic component of the nucleosome core particle, linker histone H1 directly interacts with it in a monomeric form. Histone H1 binds nucleosomes near the exit/entry sites of linker DNA, determines nucleosome repeat length and stabilizes higher-order organization of nucleosomes into the ∼30 nm chromatin fiber. In comparison to core histones, histone H1 is less well conserved through evolution. Furthermore, histone H1 composition in metazoans is generally complex with most species containing multiple variants that play redundant as well as specific functions. In this regard, a characteristic feature is the presence of specific H1 variants that replace somatic H1s in the germline and during early embryogenesis. In this review, we summarize our current knowledge about their structural and functional properties.

Computational design of co-assembling protein-DNA nanowires

NASA Astrophysics Data System (ADS)

Mou, Yun; Yu, Jiun-Yann; Wannier, Timothy M.; Guo, Chin-Lin; Mayo, Stephen L.

2015-09-01

Biomolecular self-assemblies are of great interest to nanotechnologists because of their functional versatility and their biocompatibility. Over the past decade, sophisticated single-component nanostructures composed exclusively of nucleic acids, peptides and proteins have been reported, and these nanostructures have been used in a wide range of applications, from drug delivery to molecular computing. Despite these successes, the development of hybrid co-assemblies of nucleic acids and proteins has remained elusive. Here we use computational protein design to create a protein-DNA co-assembling nanomaterial whose assembly is driven via non-covalent interactions. To achieve this, a homodimerization interface is engineered onto the Drosophila Engrailed homeodomain (ENH), allowing the dimerized protein complex to bind to two double-stranded DNA (dsDNA) molecules. By varying the arrangement of protein-binding sites on the dsDNA, an irregular bulk nanoparticle or a nanowire with single-molecule width can be spontaneously formed by mixing the protein and dsDNA building blocks. We characterize the protein-DNA nanowire using fluorescence microscopy, atomic force microscopy and X-ray crystallography, confirming that the nanowire is formed via the proposed mechanism. This work lays the foundation for the development of new classes of protein-DNA hybrid materials. Further applications can be explored by incorporating DNA origami, DNA aptamers and/or peptide epitopes into the protein-DNA framework presented here.

Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits.

PubMed

Snyder, P M; Cheng, C; Prince, L S; Rogers, J C; Welsh, M J

1998-01-09

Members of the DEG/ENaC protein family form ion channels with diverse functions. DEG/ENaC subunits associate as hetero- and homomultimers to generate channels; however the stoichiometry of these complexes is unknown. To determine the subunit stoichiometry of the human epithelial Na+ channel (hENaC), we expressed the three wild-type hENaC subunits (alpha, beta, and gamma) with subunits containing mutations that alter channel inhibition by methanethiosulfonates. The data indicate that hENaC contains three alpha, three beta, and three gamma subunits. Sucrose gradient sedimentation of alphahENaC translated in vitro, as well as alpha-, beta-, and gammahENaC coexpressed in cells, was consistent with complexes containing nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced complexes of similar mass. Our results suggest a novel nine-subunit stoichiometry for the DEG/ENaC family of ion channels.

Electrostatically assembled dendrimer complex with a high-affinity protein binder for targeted gene delivery.

PubMed

Kim, Jong-Won; Lee, Joong-Jae; Choi, Joon Sig; Kim, Hak-Sung

2018-06-10

Although a variety of non-viral gene delivery systems have been developed, they still suffer from low efficiency and specificity. Herein, we present the assembly of a dendrimer complex comprising a DNA cargo and a targeting moiety as a new format for targeted gene delivery. A PAMAM dendrimer modified with histidine and arginine (HR-dendrimer) was used to enhance the endosomal escape and transfection efficiency. An EGFR-specific repebody, composed of leucine-rich repeat (LRR) modules, was employed as a targeting moiety. A polyanionic peptide was genetically fused to the repebody, followed by incubation with an HR-dendrimer and a DNA cargo to assemble the dendrimer complex through an electrostatic interaction. The resulting dendrimer complex was shown to deliver a DNA cargo with high efficiency in a receptor-specific manner. An analysis using a confocal microscope confirmed the internalization of the dendrimer complex and subsequent dissociation of a DNA cargo from the complex. The present approach can be broadly used in a targeted gene delivery in many areas. Copyright © 2018 Elsevier B.V. All rights reserved.

Thermodynamics of Coupled Folding in the Interaction of Archaeal RNase P Proteins RPP21 and RPP29

PubMed Central

Xu, Yiren; Oruganti, Sri Vidya; Gopalan, Venkat; Foster, Mark P.

2014-01-01

We have used isothermal titration calorimetry (ITC) to identify and describe binding-coupled equilibria in the interaction between two protein subunits of archaeal ribonuclease P (RNase P). In all three domains of life, RNase P is a ribonucleoprotein complex that is primarily responsible for catalyzing the Mg2+-dependent cleavage of the 5′ leader sequence of precursor tRNAs during tRNA maturation. In archaea, RNase P has been shown to be composed of one catalytic RNA and up to five proteins, four of which associate in the absence of RNA as two functional heterodimers, POP5-RPP30 and RPP21-RPP29. NMR studies of the Pyrococcus furiosus RPP21 and RPP29 proteins in their free and complexed states provided evidence for significant protein folding upon binding. ITC experiments were performed over a range of temperatures, ionic strengths, pH values and in buffers with varying ionization potential, and with a folding-deficient RPP21 point mutant. These experiments revealed a negative heat capacity change (ΔCp), nearly twice that predicted from surface accessibility calculations, a strong salt dependence to the interaction and proton release at neutral pH, but a small net contribution from these to the excess ΔCp. We considered potential contributions from protein folding and burial of interfacial water molecules based on structural and spectroscopic data. We conclude that binding-coupled protein folding is likely responsible for a significant portion of the excess ΔCp. These findings provide novel structural-thermodynamic insights into coupled equilibria that enable specificity in macromolecular assemblies. PMID:22243443

Top-down Mass Spectrometry of Cardiac Myofilament Proteins in Health and Disease

PubMed Central

Ying, Peng; Serife, Ayaz-Guner; Deyang, Yu; Ying, Ge

2014-01-01

Myofilaments are composed of thin and thick filaments which coordinate with each other to regulate muscle contraction and relaxation. Posttranslational modifications (PTMs) together with genetic variations and alternative splicing of the myofilament proteins play essential roles in regulating cardiac contractility in health and disease. Therefore, a comprehensive characterization of the myofilament proteins in physiological and pathological conditions is essential for better understanding the molecular basis of cardiac function and dysfunction. Due to the vast complexity and dynamic nature of proteins, it is challenging to obtain a holistic view of myofilament protein modifications. In recent years, top-down mass spectrometry (MS) has emerged as a powerful approach to study isoform composition and PTMs of proteins owing to its advantage of complete sequence coverage and its ability to identify PTMs and sequence variants without a priori knowledge. In this review, we will discuss the application of top-down MS to study cardiac myofilaments and highlight the insights it provides into the understanding of molecular mechanisms in contractile dysfunction of heart failure. Particularly, recent results of cardiac troponin and tropomyosin modifications will be elaborated. The limitations and perspectives on the use of top-down MS for myofilament protein characterization will also be briefly discussed. PMID:24945106

Multidomain Carbohydrate-binding Proteins Involved in Bacteroides thetaiotaomicron Starch Metabolism*

PubMed Central

Cameron, Elizabeth A.; Maynard, Mallory A.; Smith, Christopher J.; Smith, Thomas J.; Koropatkin, Nicole M.; Martens, Eric C.

2012-01-01

Human colonic bacteria are necessary for the digestion of many dietary polysaccharides. The intestinal symbiont Bacteroides thetaiotaomicron uses five outer membrane proteins to bind and degrade starch. Here, we report the x-ray crystallographic structures of SusE and SusF, two outer membrane proteins composed of tandem starch specific carbohydrate-binding modules (CBMs) with no enzymatic activity. Examination of the two CBMs in SusE and three CBMs in SusF reveals subtle differences in the way each binds starch and is reflected in their Kd values for both high molecular weight starch and small maltooligosaccharides. Thus, each site seems to have a unique starch preference that may enable these proteins to interact with different regions of starch or its breakdown products. Proteins similar to SusE and SusF are encoded in many other polysaccharide utilization loci that are possessed by human gut bacteria in the phylum Bacteroidetes. Thus, these proteins are likely to play an important role in carbohydrate metabolism in these abundant symbiotic species. Understanding structural changes that diversify and adapt related proteins in the human gut microbial community will be critical to understanding the detailed mechanistic roles that they perform in the complex digestive ecosystem. PMID:22910908

Exploiting CELLULOSE SYNTHASE (CESA) Class Specificity to Probe Cellulose Microfibril Biosynthesis.

PubMed

Kumar, Manoj; Mishra, Laxmi; Carr, Paul; Pilling, Michael; Gardner, Peter; Mansfield, Shawn D; Turner, Simon

2018-05-01

Cellulose microfibrils are the basic units of cellulose in plants. The structure of these microfibrils is at least partly determined by the structure of the cellulose synthase complex. In higher plants, this complex is composed of 18 to 24 catalytic subunits known as CELLULOSE SYNTHASE A (CESA) proteins. Three different classes of CESA proteins are required for cellulose synthesis and for secondary cell wall cellulose biosynthesis these classes are represented by CESA4, CESA7, and CESA8. To probe the relationship between CESA proteins and microfibril structure, we created mutant cesa proteins that lack catalytic activity but retain sufficient structural integrity to allow assembly of the cellulose synthase complex. Using a series of Arabidopsis ( Arabidopsis thaliana ) mutants and genetic backgrounds, we found consistent differences in the ability of these mutant cesa proteins to complement the cellulose-deficient phenotype of the cesa null mutants. The best complementation was observed with catalytically inactive cesa4, while the equivalent mutation in cesa8 exhibited significantly lower levels of complementation. Using a variety of biophysical techniques, including solid-state nuclear magnetic resonance and Fourier transform infrared microscopy, to study these mutant plants, we found evidence for changes in cellulose microfibril structure, but these changes largely correlated with cellulose content and reflected differences in the relative proportions of primary and secondary cell walls. Our results suggest that individual CESA classes have similar roles in determining cellulose microfibril structure, and it is likely that the different effects of mutating members of different CESA classes are the consequence of their different catalytic activity and their influence on the overall rate of cellulose synthesis. © 2018 American Society of Plant Biologists. All Rights Reserved.

Exploiting CELLULOSE SYNTHASE (CESA) Class Specificity to Probe Cellulose Microfibril Biosynthesis1[OPEN

PubMed Central

Mishra, Laxmi; Carr, Paul; Gardner, Peter

2018-01-01

Cellulose microfibrils are the basic units of cellulose in plants. The structure of these microfibrils is at least partly determined by the structure of the cellulose synthase complex. In higher plants, this complex is composed of 18 to 24 catalytic subunits known as CELLULOSE SYNTHASE A (CESA) proteins. Three different classes of CESA proteins are required for cellulose synthesis and for secondary cell wall cellulose biosynthesis these classes are represented by CESA4, CESA7, and CESA8. To probe the relationship between CESA proteins and microfibril structure, we created mutant cesa proteins that lack catalytic activity but retain sufficient structural integrity to allow assembly of the cellulose synthase complex. Using a series of Arabidopsis (Arabidopsis thaliana) mutants and genetic backgrounds, we found consistent differences in the ability of these mutant cesa proteins to complement the cellulose-deficient phenotype of the cesa null mutants. The best complementation was observed with catalytically inactive cesa4, while the equivalent mutation in cesa8 exhibited significantly lower levels of complementation. Using a variety of biophysical techniques, including solid-state nuclear magnetic resonance and Fourier transform infrared microscopy, to study these mutant plants, we found evidence for changes in cellulose microfibril structure, but these changes largely correlated with cellulose content and reflected differences in the relative proportions of primary and secondary cell walls. Our results suggest that individual CESA classes have similar roles in determining cellulose microfibril structure, and it is likely that the different effects of mutating members of different CESA classes are the consequence of their different catalytic activity and their influence on the overall rate of cellulose synthesis. PMID:29523715

Pyrene-Labeled Amphiphiles: Dynamic And Structural Probes Of Membranes And Lipoproteins

NASA Astrophysics Data System (ADS)

Pownall, Henry J.; Homan, Reynold; Massey, John B.

1987-01-01

Lipids and proteins are important functional and structural components of living organisms. Although proteins are frequently found as soluble components of plasma or the cell cytoplasm, many lipids are much less soluble and separate into complex assemblies that usually contain proteins. Cell membranes and plasma lipoproteins' are two important macro-molecular assemblies that contain both lipids and proteins. Cell membranes are composed of a variety of lipids and proteins that form an insoluble bilayer array that has relatively little curvature over distances of several nm. Plasma lipoproteins are different in that they are much smaller, water-soluble, and have highly curved surfaces. A model of a high density lipoprotein (HDL) is shown in Figure 1. This model (d - 10 nm) contains a surface of polar lipids and proteins that surrounds a small core of insoluble lipids, mostly triglycerides and cholesteryl esters. The low density (LDL) (d - 25 nm) and very low density (VLDL) (d 90 nm) lipoproteins have similar architectures, except the former has a cholesteryl ester core and the latter a core that is almost exclusively triglyceride (Figure 1). The surface proteins of HDL are amphiphilic and water soluble; the single protein of LDL is insoluble, whereas VLDL contains both soluble and insoluble proteins. The primary structures of all of these proteins are known.

Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport

PubMed Central

Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

2013-01-01

Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment. PMID:23350036

Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport.

PubMed

Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

2013-01-01

Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment.

A novel TBP-TAF complex on RNA polymerase II-transcribed snRNA genes.

PubMed

Zaborowska, Justyna; Taylor, Alice; Roeder, Robert G; Murphy, Shona

2012-01-01

Initiation of transcription of most human genes transcribed by RNA polymerase II (RNAP II) requires the formation of a preinitiation complex comprising TFIIA, B, D, E, F, H and RNAP II. The general transcription factor TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors. During transcription of snRNA genes, RNAP II does not appear to make the transition to long-range productive elongation, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3' box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. For example, differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. As precedent, it already has been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5 has been shown to be associated with RNAP II-transcribed snRNA genes, the full complement of TAFs associated with these genes has remained unclear. Here we show, using a ChIP and siRNA-mediated approach, that the TBP/TAF complex on snRNA genes differs from that found on protein-coding genes. Interestingly, the largest TAF, TAF1, and the core TAFs, TAF10 and TAF4, are not detected on snRNA genes. We propose that this snRNA gene-specific TAF subset plays a key role in gene type-specific control of expression.

Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

PubMed

Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

2006-09-08

The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

Kinetic control of TolC recruitment by multidrug efflux complexes.

PubMed

Tikhonova, Elena B; Dastidar, Vishakha; Rybenkov, Valentin V; Zgurskaya, Helen I

2009-09-22

In Gram-negative pathogens, multidrug efflux pumps that provide clinically significant levels of antibiotic resistance function as three-component complexes. They are composed of the inner membrane transporters belonging to one of three superfamilies of proteins, RND, ABC, or MF; periplasmic proteins belonging to the membrane fusion protein (MFP) family; and outer membrane channels exemplified by the Escherichia coli TolC. The three-component complexes span the entire two-membrane envelope of Gram-negative bacteria and expel toxic molecules from the cytoplasmic membrane to the medium. The architecture of these complexes is expected to vary significantly because of the structural diversity of the inner membrane transporters. How the three-component pumps are assembled, their architecture, and their dynamics remain unclear. In this study, we reconstituted interactions and compared binding kinetics of the E. coli TolC with AcrA, MacA, and EmrA, the periplasmic MFPs that function in multidrug efflux with transporters from the RND, ABC, and MF superfamilies, respectively. By using surface plasmon resonance, we demonstrate that TolC interactions with MFPs are highly dynamic and sensitive to pH. The affinity of TolC to MFPs decreases in the order MacA > EmrA > AcrA. We further show that MFPs are prone to oligomerization, but differ dramatically from each other in oligomerization kinetics and stability of oligomers. The propensity of MFPs to oligomerize correlates with the stability of MFP-TolC complexes and structural features of inner membrane transporters. We propose that recruitment of TolC by various MFPs is determined not only by kinetics of MFP-TolC interactions but also by oligomerization kinetics of MFPs and pH.

Na+, K+-ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

PubMed

Hu, Yau-Chung; Chu, Keng-Fu; Yang, Wen-Kai; Lee, Tsung-Han

2017-10-01

The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na + , K + -ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and β-subunits. Among the NKA genes, α1-β1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1-β1 isozyme. Based on the sequenced milkfish transcriptome, an NKA β1 subunit gene was identified that had the highest amino acid homology to β233, a NKA β1 subunit paralog originally identified in the eel. Despite this high level of homology to β233, phylogenetic analysis and the fact that only a single NKA β1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA β1 subunit gene. The results of accurate domain prediction of the β1 subunit, co-localization of α1 and β1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and β1 subunits, indicated the formation of a α1-β1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and β1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA β1 subunit and provided the first evidence for an NKA α1-β1 association in gill ionocytes of euryhaline teleosts.

Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the Cu(A) center of cytochrome c oxidase.

PubMed

Hill, Bruce C; Andrews, Diann

2012-06-01

SCO (synthesis of cytochrome c oxidase) proteins are involved in the assembly of the respiratory chain enzyme cytochrome c oxidase acting to assist in the assembly of the Cu(A) center contained within subunit II of the oxidase complex. The Cu(A) center receives electrons from the reductive substrate ferrocytochrome c, and passes them on to the cytochrome a center. Cytochrome a feeds electrons to the oxygen reaction site composed of cytochrome a(3) and Cu(B). Cu(A) consists of two copper ions positioned within bonding distance and ligated by two histidine side chains, one methionine, a backbone carbonyl and two bridging cysteine residues. The complex structure and redox capacity of Cu(A) present a potential assembly challenge. SCO proteins are members of the thioredoxin family which led to the early suggestion of a disulfide exchange function for SCO in Cu(A) assembly, whereas the copper binding capacity of the Bacillus subtilis version of SCO (i.e., BsSCO) suggests a direct role for SCO proteins in copper transfer. We have characterized redox and copper exchange properties of apo- and metalated-BsSCO. The release of copper (II) from its complex with BsSCO is best achieved by reducing it to Cu(I). We propose a mechanism involving both disulfide and copper exchange between BsSCO and the apo-Cu(A) site. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes. Copyright © 2011 Elsevier B.V. All rights reserved.

Mitotic phosphorylation of SUN1 loosens its connection with the nuclear lamina while the LINC complex remains intact.

PubMed

Patel, Jennifer T; Bottrill, Andrew; Prosser, Suzanna L; Jayaraman, Sangeetha; Straatman, Kees; Fry, Andrew M; Shackleton, Sue

2014-01-01

At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD.

African Swine Fever Virus Gets Undressed: New Insights on the Entry Pathway.

PubMed

Andrés, Germán

2017-02-15

African swine fever virus (ASFV) is a large, multienveloped DNA virus composed of a genome-containing core successively wrapped by an inner lipid envelope, an icosahedral protein capsid, and an outer lipid envelope. In keeping with this structural complexity, recent studies have revealed an intricate entry program. This Gem highlights how ASFV uses two alternative pathways, macropinocytosis and clathrin-mediated endocytosis, to enter into the host macrophage and how the endocytosed particles undergo a stepwise, low pH-driven disassembly leading to inner envelope fusion and core delivery in the cytoplasm. Copyright © 2017 American Society for Microbiology.

Striated fibers in trichomonads: costa proteins represent a new class of proteins forming striated roots.

PubMed

Viscogliosi, E; Brugerolle, G

1994-01-01

The production of monoclonal antibodies and the use of biochemical techniques revealed that B-type costa proteins in trichomonads are composed of several major polypeptides with molecular weight detected between 100 and 135 kDa similar to those found in the A-type costae. Although differences were observed between the two types in their fine structure, we tested whether proteins composing the two costa types belong to the same protein family. A polyclonal antibody produced against the 118 kDa costa protein of Trichomonas vaginalis also recognized a 118 kDa costa protein in all other trichomonad genera studied so far whether they have A- or B-type costae. Moreover biochemical characteristics of costa proteins indicated that these proteins might represent a novel class of striated root-forming proteins in addition to centrin, giardin, and assemblin.

Rapid search for tertiary fragments reveals protein sequence–structure relationships

PubMed Central

Zhou, Jianfu; Grigoryan, Gevorg

2015-01-01

Finding backbone substructures from the Protein Data Bank that match an arbitrary query structural motif, composed of multiple disjoint segments, is a problem of growing relevance in structure prediction and protein design. Although numerous protein structure search approaches have been proposed, methods that address this specific task without additional restrictions and on practical time scales are generally lacking. Here, we propose a solution, dubbed MASTER, that is both rapid, enabling searches over the Protein Data Bank in a matter of seconds, and provably correct, finding all matches below a user-specified root-mean-square deviation cutoff. We show that despite the potentially exponential time complexity of the problem, running times in practice are modest even for queries with many segments. The ability to explore naturally plausible structural and sequence variations around a given motif has the potential to synthesize its design principles in an automated manner; so we go on to illustrate the utility of MASTER to protein structural biology. We demonstrate its capacity to rapidly establish structure–sequence relationships, uncover the native designability landscapes of tertiary structural motifs, identify structural signatures of binding, and automatically rewire protein topologies. Given the broad utility of protein tertiary fragment searches, we hope that providing MASTER in an open-source format will enable novel advances in understanding, predicting, and designing protein structure. PMID:25420575

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

PubMed Central

Brindley, Melinda A.; Plattet, Philippe; Plemper, Richard Karl

2014-01-01

Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry. PMID:25157143

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain.

PubMed

Brindley, Melinda A; Plattet, Philippe; Plemper, Richard Karl

2014-09-09

Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry.

Deletion of Smgpi1 encoding a GPI-anchored protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora.

PubMed

Frey, Stefan; Lahmann, Yasmine; Hartmann, Thomas; Seiler, Stephan; Pöggeler, Stefanie

2015-08-01

The striatin interacting phosphatase and kinase (STRIPAK) complex, which is composed of striatin, protein phosphatase PP2A and kinases, is required for fruiting-body development and cell fusion in the filamentous ascomycete Sordaria macrospora. Here, we report on the interplay of the glycosylphosphatidylinositol (GPI)-anchored protein SmGPI1 with the kinase activator SmMOB3, a core component of human and fungal STRIPAK complexes. SmGPI1 is conserved among filamentous ascomycetes and was first identified in a yeast two-hybrid screen using SmMOB3 as bait. The physical interaction of SmMOB3 and SmGPI1 was verified by co-immunoprecipitation. In vivo localization and differential centrifugation revealed that SmGPI1 is predominantly secreted and attached to the cell wall but is also associated with mitochondria and appears to be a dual-targeted protein. Deletion of Smgpi1 led to an increased number of fruiting bodies that were normally shaped but reduced in size. In addition, Smmob3 and Smgpi1 genetically interact. In the sterile ΔSmmob3 background deletion of Smgpi1 restores fertility, vegetative growth as well as hyphal-fusion defects. The suppression effect was specific for the ΔSmmob3 mutant as deletion of Smgpi1 in other STRIPAK mutants does not restore fertility. © 2015 John Wiley & Sons Ltd.

Signaling pathways that regulate life and cell death: evolution of apoptosis in the context of self-defense.

PubMed

Muñoz-Pinedo, Cristina

2012-01-01

Programmed Cell Death is essential for the life cycle of many organisms. Cell death in multicellular organisms can occur as a consequence of massive damage (necrosis) or in a controlled form, through engagement of diverse biochemical programs. The best well known form of programmed cell death is apoptosis. Apoptosis occurs in animals as a consequence of a variety of stimuli including stress and social signals and it plays essential roles in morphogenesis and immune defense. The machinery of apoptosis is well conserved among animals and it is composed of caspases (the proteases which execute cell death), adapter proteins (caspase activators), Bcl-2 family proteins and Inhibitor of Apoptosis Proteins (IAPs). We will describe in this chapter the main apoptotic pathways in animals: the extrinsic (death receptor-mediated), the intrinsic/mitochondrial and the Granzyme B pathway. Other forms of non-apoptotic Programmed Cell Death which occur in animals will also be discussed. We will summarize the current knowledge about apoptotic-like and other forms of cell death in other organisms such as plants and protists.Additionally, we will discuss the hypothesis that apoptosis originated as part of a host defense mechanism. We will explore the similarities between the protein complexes which mediate apoptosis (apoptosomes) and complexes involved in immunity: inflammasomes. Additional functions of apoptotic proteins related to immune function will be summarized, in an effort to explore the evolutionary origins of cell death.

Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery

PubMed Central

Maio, Nunziata; Rouault, Tracey. A.

2014-01-01

Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i. e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein- protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. PMID:25245479

Ethanol and Protein from Ethanol Plant By-Products Using Edible Fungi Neurospora intermedia and Aspergillus oryzae

PubMed Central

Bátori, Veronika; Ferreira, Jorge A.; Taherzadeh, Mohammad J.; Lennartsson, Patrik R.

2015-01-01

Feasible biorefineries for production of second-generation ethanol are difficult to establish due to the process complexity. An alternative is to partially include the process in the first-generation plants. Whole stillage, a by-product from dry-mill ethanol processes from grains, is mostly composed of undegraded bran and lignocelluloses can be used as a potential substrate for production of ethanol and feed proteins. Ethanol production and the proteins from the stillage were investigated using the edible fungi Neurospora intermedia and Aspergillus oryzae, respectively. N. intermedia produced 4.7 g/L ethanol from the stillage and increased to 8.7 g/L by adding 1 FPU of cellulase/g suspended solids. Saccharomyces cerevisiae produced 0.4 and 5.1 g/L ethanol, respectively. Under a two-stage cultivation with both fungi, up to 7.6 g/L of ethanol and 5.8 g/L of biomass containing 42% (w/w) crude protein were obtained. Both fungi degraded complex substrates including arabinan, glucan, mannan, and xylan where reductions of 91, 73, 38, and 89% (w/v) were achieved, respectively. The inclusion of the current process can lead to the production of 44,000 m3 of ethanol (22% improvement), around 12,000 tons of protein-rich biomass for animal feed, and energy savings considering a typical facility producing 200,000 m3 ethanol/year. PMID:26682213

Selective complexation of K+ and Na+ in simple polarizable ion-ligating systems.

PubMed

Bostick, David L; Brooks, Charles L

2010-09-29

An influx of experimental and theoretical studies of ion transport protein structure has inspired efforts to understand underlying determinants of ionic selectivity. Design principles for selective ion binding can be effectively isolated and interrogated using simplified models composed of a single ion surrounded by a set of ion-ligating molecular species. While quantum mechanical treatments of such systems naturally incorporate electronic degrees of freedom, their computational overhead typically prohibits thorough dynamic sampling of configurational space and, thus, requires approximations when determining ion-selective free energy. As an alternative, we employ dynamical simulations with a polarizable force field to probe the structure and K(+)/Na(+) selectivity in simple models composed of one central K(+)/Na(+) ion surrounded by 0-8 identical model compounds: N-methylacetamide, formamide, or water. In the absence of external restraints, these models represent gas-phase clusters displaying relaxed coordination structures with low coordination number. Such systems display Na(+) selectivity when composed of more than ∼3 organic carbonyl-containing compounds and always display K(+) selectivity when composed of water molecules. Upon imposing restraints that solely enforce specific coordination numbers, we find all models are K(+)-selective when ∼7-8-fold ion coordination is achieved. However, when models composed of the organic compounds provide ∼4-6-fold coordination, they retain their Na(+) selectivity. From these trends, design principles emerge that are of basic importance in the behavior of K(+) channel selectivity filters and suggest a basis not only for K(+) selectivity but also for modulation of block and closure by smaller ions.

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

Zhang, Yanfeng; Zheng, Yi; Qin, Ling

Beta-hydroxyacid dehydrogenase (β-HAD) genes have been identified in all sequenced genomes of eukaryotes and prokaryotes. Their gene products catalyze the NAD+- or NADP+-dependent oxidation of various β-hydroxy acid substrates into their corresponding semialdehyde. In many fungal and bacterial genomes, multiple β-HAD genes are observed leading to the hypothesis that these gene products may have unique, uncharacterized metabolic roles specific to their species. The genomes of Geobacter sulfurreducens and Geobacter metallireducens each contain two potential β-HAD genes. The protein sequences of one pair of these genes, Gs-βHAD (Q74DE4) and Gm-βHAD (Q39R98), have 65% sequence identity and 77% sequence similarity with eachmore » other. Both proteins reduce succinic semialdehyde, a metabolite of the GABA shunt. To further explore the structural and functional characteristics of these two β-HADs with a potentially unique substrate specificity, crystal structures for Gs-βHAD and Gm-βHAD in complex with NADP+ were determined to a resolution of 1.89 Å and 2.07 Å, respectively. The structure of both proteins are similar, composed of 14 α-helices and nine β-strands organized into two domains. Domain One (1-165) adopts a typical Rossmann fold composed of two α/β units: a six-strand parallel β-sheet surrounded by six α-helices (α1 – α6) followed by a mixed three-strand β-sheet surrounded by two α-helices (α7 and α8). Domain Two (166-287) is composed of a bundle of seven α-helices (α9 – α14). Four functional regions conserved in all β-HADs are spatially located near each other at the interdomain cleft in both Gs-βHAD and Gm-βHAD with a buried molecule of NADP+. The structural features of Gs-βHAD and Gm-βHAD are described in relation to the four conserved consensus sequences characteristic of β-HADs and the potential biochemical importance of these enzymes as an alternative pathway for the degradation of succinic semialdehyde.« less

Selective extraction and enrichment of multiphosphorylated peptides using polyarginine-coated diamond nanoparticles.

PubMed

Chang, Chia-Kai; Wu, Chih-Che; Wang, Yi-Sheng; Chang, Huan-Cheng

2008-05-15

Despite recent advances in phosphopeptide research, detection and characterization of multiply phosphorylated peptides have been a challenge. This work presents a new strategy that not only can effectively extract phosphorylated peptides from complex samples but also can selectively enrich multiphosphorylated peptides for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Polyarginine-coated diamond nanoparticles are the solid-phase extraction supports used for this purpose. The supports show an exceptionally high affinity for multiphosphorylated peptides due to multiple arginine-phosphate interactions. The efficacy of this method was demonstrated by analyzing a small volume (50 microL) of tryptic digests of proteins such as beta-casein, alpha-casein, and nonfat milk at a concentration as low as 1 x 10 (-9) M. The concentration is markedly lower than that can be achieved by using other currently available technologies. We quantified the enhanced selectivity and detection sensitivity of the method using mixtures composed of mono- and tetraphosphorylated peptide standards. This new affinity-based protocol is expected to find useful applications in characterizing multiple phosphorylation sites on proteins of interest in complex and dilute analytes.

Crystal Structure of the Ternary Complex of a NaV C-Terminal Domain, a Fibroblast Growth Factor Homologous Factor, and Calmodulin

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

Wang, Chaojian; Chung, Ben C.; Yan, Haidun

2012-11-13

Voltage-gated Na{sup +} (Na{sub V}) channels initiate neuronal action potentials. Na{sub V} channels are composed of a transmembrane domain responsible for voltage-dependent Na{sup +} conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins, including fibroblast growth factor homologous factors (FHFs) and calmodulin (CaM). Most ion channel structural studies have focused on mechanisms of permeation and voltage-dependent gating but less is known about how intracellular domains modulate channel function. Here we report the crystal structure of the ternary complex of a human NaV CTD, an FHF, and Ca{sup 2+}-free CaM at 2.2 {angstrom}.more » Combined with functional experiments based on structural insights, we present a platform for understanding the roles of these auxiliary proteins in NaV channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity of individual NaV CTD isoforms for distinctive FHFs.« less

The epilepsy-linked Lgi1 protein assembles into presynaptic Kv1 channels and inhibits inactivation by Kvbeta1.

PubMed

Schulte, Uwe; Thumfart, Jörg-Oliver; Klöcker, Nikolaj; Sailer, Claudia A; Bildl, Wolfgang; Biniossek, Martin; Dehn, Doris; Deller, Thomas; Eble, Silke; Abbass, Karen; Wangler, Tanja; Knaus, Hans-Günther; Fakler, Bernd

2006-03-02

The voltage-gated potassium (Kv) channel subunit Kv1.1 is a major constituent of presynaptic A-type channels that modulate synaptic transmission in CNS neurons. Here, we show that Kv1.1-containing channels are complexed with Lgi1, the functionally unassigned product of the leucine-rich glioma inactivated gene 1 (LGI1), which is causative for an autosomal dominant form of lateral temporal lobe epilepsy (ADLTE). In the hippocampal formation, both Kv1.1 and Lgi1 are coassembled with Kv1.4 and Kvbeta1 in axonal terminals. In A-type channels composed of these subunits, Lgi1 selectively prevents N-type inactivation mediated by the Kvbeta1 subunit. In contrast, defective Lgi1 molecules identified in ADLTE patients fail to exert this effect resulting in channels with rapid inactivation kinetics. The results establish Lgi1 as a novel subunit of Kv1.1-associated protein complexes and suggest that changes in inactivation gating of presynaptic A-type channels may promote epileptic activity.

NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO

PubMed Central

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Mueller, Alexander P.; Köpke, Michael

2013-01-01

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP+ with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0′ = −520 mV). PMID:23893107

NADP-specific electron-bifurcating [FeFe]-hydrogenase in a functional complex with formate dehydrogenase in Clostridium autoethanogenum grown on CO.

PubMed

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Mueller, Alexander P; Köpke, Michael; Thauer, Rudolf K

2013-10-01

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP(+) with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0' = -520 mV).

Kinetics of the formation of a protein corona around nanoparticles.

PubMed

Zhdanov, Vladimir P; Cho, Nam-Joon

2016-12-01

Interaction of metal or oxide nanoparticles (NPs) with biological soft matter is one of the central phenomena in basic and applied biology-oriented nanoscience. Often, this interaction includes adsorption of suspended proteins on the NP surface, resulting in the formation of the protein corona around NPs. Structurally, the corona contains a "hard" monolayer shell directly contacting a NP and a more distant weakly associated "soft" shell. Chemically, the corona is typically composed of a mixture of distinct proteins. The corresponding experimental and theoretical studies have already clarified many aspects of the corona formation. The process is, however, complex, and its understanding is still incomplete. Herein, we present a kinetic mean-field model of the formation of the "hard" corona with emphasis on the role of (i) protein-diffusion limitations and (ii) interplay between competitive adsorption of distinct proteins and irreversible reconfiguration of their native structure. The former factor is demonstrated to be significant only in the very beginning of the corona formation. The latter factor is predicted to be more important. It may determine the composition of the corona on the time scales comparable or longer than a few hours. Copyright © 2016 Elsevier Inc. All rights reserved.

SERS Detection of Amyloid Oligomers on Metallorganic-Decorated Plasmonic Beads.

PubMed

Guerrini, Luca; Arenal, Raul; Mannini, Benedetta; Chiti, Fabrizio; Pini, Roberto; Matteini, Paolo; Alvarez-Puebla, Ramon A

2015-05-13

Protein misfolded proteins are among the most toxic endogenous species of macromolecules. These chemical entities are responsible for neurodegenerative disorders such as Alzheimer's, Parkinson's, Creutzfeldt-Jakob's and different non-neurophatic amyloidosis. Notably, these oligomers show a combination of marked heterogeneity and low abundance in body fluids, which have prevented a reliable detection by immunological methods so far. Herein we exploit the selectivity of proteins to react with metallic ions and the sensitivity of surface-enhanced Raman spectroscopy (SERS) toward small electronic changes in coordination compounds to design and engineer a reliable optical sensor for protein misfolded oligomers. Our strategy relies on the functionalization of Au nanoparticle-decorated polystyrene beads with an effective metallorganic Raman chemoreceptor, composed by Al(3+) ions coordinated to 4-mercaptobenzoic acid (MBA) with high Raman cross-section, that selectively binds aberrant protein oligomers. The mechanical deformations of the MBA phenyl ring upon complexation with the oligomeric species are registered in its SERS spectrum and can be quantitatively correlated with the concentration of the target biomolecule. The SERS platform used here appears promising for future implementation of diagnostic tools of aberrant species associated with protein deposition diseases, including those with a strong social and economic impact, such as Alzheimer's and Parkinson's diseases.

Energy-converting [NiFe] hydrogenases from archaea and extremophiles: ancestors of complex I.

PubMed

Hedderich, Reiner

2004-02-01

[NiFe] hydrogenases are well-characterized enzymes that have a key function in the H2 metabolism of various microorganisms. In the recent years a subfamily of [NiFe] hydrogenases with unique properties has been identified. The members of this family form multisubunit membrane-bound enzyme complexes composed of at least four hydrophilic and two integral membrane proteins. These six conserved subunits, which built the core of these hydrogenases, have closely related counterparts in energy-conserving NADH:quinone oxidoreductases (complex I). However, the reaction catalyzed by these hydrogenases differs significantly from the reaction catalyzed by complex I. For some of these hydrogenases the physiological role is to catalyze the reduction of H+ with electrons derived from reduced ferredoxins or poly-ferredoxins. This exergonic reaction is coupled to energy conservation by means of electron-transport phosphorylation. Other members of this hydrogenase family mainly function to provide the cell with reduced ferredoxin with H2 as electron donor in a reaction driven by reverse electron transport. As complex I these hydrogenases function as ion pumps and have therefore been designated as energy-converting [NiFe] hydrogenases.

Subcutaneous immunization with inactivated bacterial components and purified protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes prevents puerperal metritis in Holstein dairy cows.

PubMed

Machado, Vinícius Silva; Bicalho, Marcela Luccas de Souza; Meira Junior, Enoch Brandão de Souza; Rossi, Rodolfo; Ribeiro, Bruno Leonardo; Lima, Svetlana; Santos, Thiago; Kussler, Arieli; Foditsch, Carla; Ganda, Erika Korzune; Oikonomou, Georgios; Cheong, Soon Hon; Gilbert, Robert Owen; Bicalho, Rodrigo Carvalho

2014-01-01

In this study we evaluate the efficacy of five vaccine formulations containing different combinations of proteins (FimH; leukotoxin, LKT; and pyolysin, PLO) and/or inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes) in preventing postpartum uterine diseases. Inactivated whole cells were produced using two genetically distinct strains of each bacterial species (E. coli, F. necrophorum, and T. pyogenes). FimH and PLO subunits were produced using recombinant protein expression, and LKT was recovered from culturing a wild F. necrophorum strain. Three subcutaneous vaccines were formulated: Vaccine 1 was composed of inactivated bacterial whole cells and proteins; Vaccine 2 was composed of proteins only; and Vaccine 3 was composed of inactivated bacterial whole cells only. Two intravaginal vaccines were formulated: Vaccine 4 was composed of inactivated bacterial whole cells and proteins; and Vaccine 5 was composed of PLO and LKT. To evaluate vaccine efficacy, a randomized clinical trial was conducted at a commercial dairy farm; 371 spring heifers were allocated randomly into one of six different treatments groups: control, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifers assigned to one of the vaccine groups were each vaccinated twice: at 230 and 260 days of pregnancy. When vaccines were evaluated grouped as subcutaneous and intravaginal, the subcutaneous ones were found to significantly reduce the incidence of puerperal metritis. Additionally, subcutaneous vaccination significantly reduced rectal temperature at 6±1 days in milk. Reproduction was improved for cows that received subcutaneous vaccines. In general, vaccination induced a significant increase in serum IgG titers against all antigens, with subcutaneous vaccination again being more effective. In conclusion, subcutaneous vaccination with inactivated bacterial components and/or protein subunits of E. coli, F. necrophorum and T. pyogenes can prevent puerperal metritis during the first lactation of dairy cows, leading to improved reproduction.

Selecting for Fast Protein-Protein Association As Demonstrated on a Random TEM1 Yeast Library Binding BLIP.

PubMed

Cohen-Khait, Ruth; Schreiber, Gideon

2018-04-27

Protein-protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein-protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-β-lactamase library against its natural nanomolar affinity binder β-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.

Characterization of the Catalytic Subunits of the RNA Exosome-like Complex in Plasmodium falciparum.

PubMed

Jiang, Ning; Yu, Shengchao; Yang, Na; Feng, Ying; Sang, Xiaoyu; Wang, Yao; Wahlgren, Mats; Chen, Qijun

2018-04-17

The eukaryotic ribonucleic acid (RNA) exosome is a versatile multiribonuclease complex that mediates the processing, surveillance, and degradation of virtually all classes of RNA in both the nucleus and cytoplasm. The complex, composed of 10 to 11 subunits, has been widely described in many organisms. Bioinformatic analyses revealed that there may be also an exosome-like complex in Plasmodium falciparum, a parasite of great importance in public health, with eight predicted subunits having high sequence similarity to their counterparts in yeast and human. In this work, the putative RNA catalytic components, designated as PfRrp4, PfRrp41, PfDis3, and PfRrp6, were identified and systematically analyzed. Quantitative polymerase chain reaction (QPCR) analyses suggested that all of them were transcribed steadily throughout the asexual stage. The expression of these proteins was determined by Western blot, and their localization narrowed to the cytoplasm of the parasite by indirect immunofluorescence. The recombinant proteins of PfRrp41, PfDis3, and PfRrp6 exhibited catalytic activity for single-stranded RNA (ssRNA), whereas PfRrp4 showed no processing activity of both ssRNA and dsRNA. The identification of these putative components of the RNA exosome complex opens up new perspectives for a deep understanding of RNA metabolism in the malarial parasite P. falciparum. © 2018 The Authors Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes.

PubMed

Balasubramaniam, Muthukumar; Davids, Benem; Addai, Amma B; Pandhare, Jui; Dash, Chandravanu

2017-02-22

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3' ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Chaperoning 5S RNA assembly

PubMed Central

Madru, Clément; Lebaron, Simon; Blaud, Magali; Delbos, Lila; Pipoli, Juliana; Pasmant, Eric; Réty, Stéphane; Leulliot, Nicolas

2015-01-01

In eukaryotes, three of the four ribosomal RNAs (rRNAs)—the 5.8S, 18S, and 25S/28S rRNAs—are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2–Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2–Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2–Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit. PMID:26159998

Redundant role of tissue-selective TAF(II)105 in B lymphocytes.

PubMed

Freiman, Richard N; Albright, Shane R; Chu, Leslie E; Zheng, Shuang; Liang, Hong-Erh; Sha, William C; Tjian, Robert

2002-09-01

Regulated gene expression is a complex process achieved through the function of multiple protein factors acting in concert at a given promoter. The transcription factor TFIID is a central component of the machinery regulating mRNA synthesis by RNA polymerase II. This large multiprotein complex is composed of the TATA box binding protein (TBP) and several TBP-associated factors (TAF(II)s). The recent discovery of multiple TBP-related factors and tissue-specific TAF(II)s suggests the existence of specialized TFIID complexes that likely play a critical role in regulating transcription in a gene- and tissue-specific manner. The tissue-selective factor TAF(II)105 was originally identified as a component of TFIID derived from a human B-cell line. In this report we demonstrate the specific induction of TAF(II)105 in cultured B cells in response to bacterial lipopolysaccharide (LPS). To examine the in vivo role of TAF(II)105, we have generated TAF(II)105-null mice by homologous recombination. Here we show that B-lymphocyte development is largely unaffected by the absence of TAF(II)105. TAF(II)105-null B cells can proliferate in response to LPS, produce relatively normal levels of resting antibodies, and can mount an immune response by producing antigen-specific antibodies in response to immunization. Taken together, we conclude that the function of TAF(II)105 in B cells is likely redundant with the function of other TAF(II)105-related cellular proteins.

Distinct mutations in yeast TAF(II)25 differentially affect the composition of TFIID and SAGA complexes as well as global gene expression patterns.

PubMed

Kirschner, Doris B; vom Baur, Elmar; Thibault, Christelle; Sanders, Steven L; Gangloff, Yann-Gaël; Davidson, Irwin; Weil, P Anthony; Tora, Làszlò

2002-05-01

The RNA polymerase II transcription factor TFIID, composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s), nucleates preinitiation complex formation at protein-coding gene promoters. SAGA, a second TAF(II)-containing multiprotein complex, is involved in transcription regulation in Saccharomyces cerevisiae. One of the essential protein components common to SAGA and TFIID is yTAF(II)25. We define a minimal evolutionarily conserved 91-amino-acid region of TAF(II)25 containing a histone fold domain that is necessary and sufficient for growth in vivo. Different temperature-sensitive mutations of yTAF(II)25 or chimeras with the human homologue TAF(II)30 arrested cell growth at either the G(1) or G(2)/M cell cycle phase and displayed distinct phenotypic changes and gene expression patterns. Immunoprecipitation studies revealed that TAF(II)25 mutation-dependent gene expression and phenotypic changes correlated at least partially with the integrity of SAGA and TFIID. Genome-wide expression analysis revealed that the five TAF(II)25 temperature-sensitive mutant alleles individually affect the expression of between 18 and 33% of genes, whereas taken together they affect 64% of all class II genes. Thus, different yTAF(II)25 mutations induce distinct phenotypes and affect the regulation of different subsets of genes, demonstrating that no individual TAF(II) mutant allele reflects the full range of its normal functions.

Identification of a domain within human TAF(I)48, a subunit of Selectivity Factor 1, that interacts with helix 2 of TBP.

PubMed

Xu, Shuping; Hori, Roderick T

2004-09-01

RNA polymerase I transcription in human cells requires Selectivity Factor 1, a multisubunit complex composed of the TATA-box-binding protein (TBP) and three TBP-associated factors (TAFs) called TAF(I)48, TAF(I)63 and TAF(I)110. Each of the Selectivity Factor 1 subunits binds directly to the other three components, but these interactions have not been characterized. This study is the initial identification and analysis of a TBP-binding domain within a Selectivity Factor 1 TAF. The interaction between human TBP and human TAF(I)48 was initially examined using the yeast two-hybrid assay, and a TBP-binding domain was identified in the carboxyl-terminus of human (h)TAF(I)48. Consistent with this result, the hTAF(I)48 carboxyl-terminus was able to bind directly to TBP in protein-protein interaction assays. When mutations were introduced into the hTAF(I)48 carboxyl-terminus, we identified changes in uncharged and positive residues that affect its interaction with TBP. By examining TBP mutants, residues within and adjacent to helix 2 of TBP, previously demonstrated to interact with subunits of other TBP-containing complexes [Transcription Factor IID (TFIID) and TFIIIB] were also found to diminish its affinity for the carboxyl-terminus of hTAF(I)48. The regions of hTAF(I)48 and TBP that interact are compared to those identified within other complexes containing TBP.

Pulmonary immunity and extracellular matrix interactions.

PubMed

O'Dwyer, David N; Gurczynski, Stephen J; Moore, Bethany B

2018-04-09

The lung harbors a complex immune system composed of both innate and adaptive immune cells. Recognition of infection and injury by receptors on lung innate immune cells is crucial for generation of antigen-specific responses by adaptive immune cells. The extracellular matrix of the lung, comprising the interstitium and basement membrane, plays a key role in the regulation of these immune systems. The matrix consists of several hundred assembled proteins that interact to form a bioactive scaffold. This template, modified by enzymes, acts to facilitate cell function and differentiation and changes dynamically with age and lung disease. Herein, we explore relationships between innate and adaptive immunity and the lung extracellular matrix. We discuss the interactions between extracellular matrix proteins, including glycosaminoglycans, with prominent effects on innate immune signaling effectors such as toll-like receptors. We describe the relationship of extracellular matrix proteins with adaptive immunity and leukocyte migration to sites of injury within the lung. Further study of these interactions will lead to greater knowledge of the role of matrix biology in lung immunity. The development of novel therapies for acute and chronic lung disease is dependent on a comprehensive understanding of these complex matrix-immunity interactions. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Evaluation of capillary zone electrophoresis for the quality control of complex biologic samples: Application to snake venoms.

PubMed

Kpaibe, André P S; Ben-Ameur, Randa; Coussot, Gaëlle; Ladner, Yoann; Montels, Jérôme; Ake, Michèle; Perrin, Catherine

2017-08-01

Snake venoms constitute a very promising resource for the development of new medicines. They are mainly composed of very complex peptide and protein mixtures, which composition may vary significantly from batch to batch. This latter consideration is a challenge for routine quality control (QC) in the pharmaceutical industry. In this paper, we report the use of capillary zone electrophoresis for the development of an analytical fingerprint methodology to assess the quality of snake venoms. The analytical fingerprint concept is being widely used for the QC of herbal drugs but rarely for venoms QC so far. CZE was chosen for its intrinsic efficiency in the separation of protein and peptide mixtures. The analytical fingerprint methodology was first developed and evaluated for a particular snake venom, Lachesis muta. Optimal analysis conditions required the use of PDADMAC capillary coating to avoid protein and peptide adsorption. Same analytical conditions were then applied to other snake venom species. Different electrophoretic profiles were obtained for each venom. Excellent repeatability and intermediate precision was observed for each batch. Analysis of different batches of the same species revealed inherent qualitative and quantitative composition variations of the venoms between individuals. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interaction of Vascular Smooth Muscle Cells Under Low Shear Stress

NASA Technical Reports Server (NTRS)

Seidel, Charles L.

1998-01-01

The blood vessel wall consists of three cellular layers, an outer adventitial, a middle medial and an inner intimal layer. When the blood vessel forms in the embryo it begins as a tube composed of a single cell type called endothelial cells. Over time, other cells are recruited from the surrounding tissue to form additional layers on the outer surface of the endothelial tube. The cells that are recruited are called mesenchymal cells. Mesenchymal cells are responsible for the production of connective tissue that holds the blood vessel together and for developing into vascular smooth muscle cells that are responsible for regulating the diameter of the vessel (1) and therefore, blood flow. In a fully developed blood vessel, the endothelial cells make- up the majority of cells in the intimal layer while the mesenchymal cells make-up the majority of cells in the medial and adventitial layers. Within the medial layer of a mature vessel, cells are organized into multiple circular layers of alternating bands of connective tissue and cells. The cell layer is composed of a mixture of mesenchymal cells that have not developed into smooth muscle cells and fully developed smooth muscle cells (2). The assembly and organization of complex tissues is directed in part by a signaling system composed of proteins on the cell surface called adhesion molecules. Adhesion molecules enable cells to recognize each other as well as the composition of the connective tissue in which they reside (3). It was hypothesized that the different cell types that compose the vascular wall possess different adhesion molecules that enable them to recognize each other and through this recognition system, form the complex layered organization of the vascular wall. In other words, the layered organization is an intrinsic property of the cells. If this hypothesis is correct then the different cells that make up the vessel wall, when mixed together, should organize themselves into a layered structure resembling an intact blood vessel. Experiments described below were designed to test this hypothesis.

Roles of mTOR Signaling in Brain Development.

PubMed

Lee, Da Yong

2015-09-01

mTOR is a serine/threonine kinase composed of multiple protein components. Intracellular signaling of mTOR complexes is involved in many of physiological functions including cell survival, proliferation and differentiation through the regulation of protein synthesis in multiple cell types. During brain development, mTOR-mediated signaling pathway plays a crucial role in the process of neuronal and glial differentiation and the maintenance of the stemness of neural stem cells. The abnormalities in the activity of mTOR and its downstream signaling molecules in neural stem cells result in severe defects of brain developmental processes causing a significant number of brain disorders, such as pediatric brain tumors, autism, seizure, learning disability and mental retardation. Understanding the implication of mTOR activity in neural stem cells would be able to provide an important clue in the development of future brain developmental disorder therapies.

Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

PubMed

Lee, Sukyeong; Augustin, Steffen; Tatsuta, Takashi; Gerdes, Florian; Langer, Thomas; Tsai, Francis T F

2011-02-11

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

Writing DNA with GenoCAD.

PubMed

Czar, Michael J; Cai, Yizhi; Peccoud, Jean

2009-07-01

Chemical synthesis of custom DNA made to order calls for software streamlining the design of synthetic DNA sequences. GenoCAD (www.genocad.org) is a free web-based application to design protein expression vectors, artificial gene networks and other genetic constructs composed of multiple functional blocks called genetic parts. By capturing design strategies in grammatical models of DNA sequences, GenoCAD guides the user through the design process. By successively clicking on icons representing structural features or actual genetic parts, complex constructs composed of dozens of functional blocks can be designed in a matter of minutes. GenoCAD automatically derives the construct sequence from its comprehensive libraries of genetic parts. Upon completion of the design process, users can download the sequence for synthesis or further analysis. Users who elect to create a personal account on the system can customize their workspace by creating their own parts libraries, adding new parts to the libraries, or reusing designs to quickly generate sets of related constructs.

The Regulation of Coenzyme Q Biosynthesis in Eukaryotic Cells: All That Yeast Can Tell Us

PubMed Central

González-Mariscal, Isabel; García-Testón, Elena; Padilla, Sergio; Martín-Montalvo, Alejandro; Pomares Viciana, Teresa; Vazquez-Fonseca, Luis; Gandolfo Domínguez, Pablo; Santos-Ocaña, Carlos

2014-01-01

Coenzyme Q (CoQ) is a mitochondrial lipid, which functions mainly as an electron carrier from complex I or II to complex III at the mitochondrial inner membrane, and also as antioxidant in cell membranes. CoQ is needed as electron acceptor in β-oxidation of fatty acids and pyridine nucleotide biosynthesis, and it is responsible for opening the mitochondrial permeability transition pore. The yeast model has been very useful to analyze the synthesis of CoQ, and therefore, most of the knowledge about its regulation was obtained from the Saccharomyces cerevisiae model. CoQ biosynthesis is regulated to support 2 processes: the bioenergetic metabolism and the antioxidant defense. Alterations of the carbon source in yeast, or in nutrient availability in yeasts or mammalian cells, upregulate genes encoding proteins involved in CoQ synthesis. Oxidative stress, generated by chemical or physical agents or by serum deprivation, modifies specifically the expression of some COQ genes by means of stress transcription factors such as Msn2/4p, Yap1p or Hsf1p. In general, the induction of COQ gene expression produced by metabolic changes or stress is modulated downstream by other regulatory mechanisms such as the protein import to mitochondria, the assembly of a multi-enzymatic complex composed by Coq proteins and also the existence of a phosphorylation cycle that regulates the last steps of CoQ biosynthesis. The CoQ biosynthetic complex assembly starts with the production of a nucleating lipid such as HHB by the action of the Coq2 protein. Then, the Coq4 protein recognizes the precursor HHB acting as the nucleus of the complex. The activity of Coq8p, probably as kinase, allows the formation of an initial pre-complex containing all Coq proteins with the exception of Coq7p. This pre-complex leads to the synthesis of 5-demethoxy-Q6 (DMQ6), the Coq7p substrate. When de novo CoQ biosynthesis is required, Coq7p becomes dephosphorylated by the action of Ptc7p increasing the synthesis rate of CoQ6. This critical model is needed for a better understanding of CoQ biosynthesis. Taking into account that patients with CoQ10 deficiency maintain to some extent the machinery to synthesize CoQ, new promising strategies for the treatment of CoQ10 deficiency will require a better understanding of the regulation of CoQ biosynthesis in the future. PMID:25126044

Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes.

PubMed

Book, Adam J; Gladman, Nicholas P; Lee, Sang-Sook; Scalf, Mark; Smith, Lloyd M; Vierstra, Richard D

2010-08-13

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes*

PubMed Central

Book, Adam J.; Gladman, Nicholas P.; Lee, Sang-Sook; Scalf, Mark; Smith, Lloyd M.; Vierstra, Richard D.

2010-01-01

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis. PMID:20516081

Maintenance of tumor initiating cells of defined genetic composition by nucleostemin.

PubMed

Okamoto, Naoko; Yasukawa, Mami; Nguyen, Christine; Kasim, Vivi; Maida, Yoshiko; Possemato, Richard; Shibata, Tatsuhiro; Ligon, Keith L; Fukami, Kiyoko; Hahn, William C; Masutomi, Kenkichi

2011-12-20

Recent work has identified a subset of cells resident in tumors that exhibit properties similar to those found in normal stem cells. Such cells are highly tumorigenic and may be involved in resistance to treatment. However, the genes that regulate the tumor initiating cell (TIC) state are unknown. Here, we show that overexpression of either of the nucleolar GTP-binding proteins nucleostemin (NS) or GNL3L drives the fraction of genetically defined tumor cells that exhibit markers and tumorigenic properties of TICs. Specifically, cells that constitutively express elevated levels of NS or GNL3L exhibit increased TWIST expression, phosphorylation of STAT3, expression of genes that induce pluripotent stem cells, and enhanced radioresistance; in addition, they form tumors even when small numbers of cells are implanted and exhibit an increased propensity to metastasize. GNL3L/NS forms a complex with the telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] and the SWItch-Sucrose NonFermentable (SWI-SNF) complex protein brahma-related gene 1 (BRG1), and the expression of each of these components is necessary to facilitate the cancer stem cell state. Together, these observations define a complex composed of TERT, BRG1, and NS/GNL3L that maintains the function of TICs.

Venture from the Interior-Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane.

PubMed

Bailer, Susanne M.

2017-11-25

Herpesviral capsid assembly is initiated in the nucleoplasm of the infected cell. Size constraints require that newly formed viral nucleocapsids leave the nucleus by an evolutionarily conserved vescular transport mechanism called nuclear egress. Mature capsids released from the nucleoplasm are engaged in a membrane-mediated budding process, composed of primary envelopment at the inner nuclear membrane and de-envelopment at the outer nuclear membrane. Once in the cytoplasm, the capsids receive their secondary envelope for maturation into infectious virions. Two viral proteins conserved throughout the herpesvirus family, the integral membrane protein pUL34 and the phosphoprotein pUL31, form the nuclear egress complex required for capsid transport from the infected nucleus to the cytoplasm. Formation of the nuclear egress complex results in budding of membrane vesicles revealing its function as minimal virus-encoded membrane budding and scission machinery. The recent structural analysis unraveled details of the heterodimeric nuclear egress complex and the hexagonal coat it forms at the inside of budding vesicles to drive primary envelopment. With this review, I would like to present the capsid-escort-model where pUL31 associates with capsids in nucleoplasmic replication compartments for escort to sites of primary envelopment thereby coupling capsid maturation and nuclear egress.

PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3.

PubMed

Noh, Hae Sook; Hah, Young-Sool; Zada, Sahib; Ha, Ji Hye; Sim, Gyujin; Hwang, Jin Seok; Lai, Trang Huyen; Nguyen, Huynh Quoc; Park, Jae-Yong; Kim, Hyun Joon; Byun, June-Ho; Hahm, Jong Ryeal; Kang, Kee Ryeon; Kim, Deok Ryong

2016-11-01

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 β) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.

Nucleoporin MOS7/Nup88 contributes to plant immunity and nuclear accumulation of defense regulators.

PubMed

Wiermer, Marcel; Germain, Hugo; Cheng, Yu Ti; García, Ana V; Parker, Jane E; Li, Xin

2010-01-01

Controlled nucleocytoplasmic trafficking is an important feature for fine-tuning signaling pathways in eukaryotic organisms. Nuclear pore complexes (NPCs) composed of nucleoporin proteins (Nups) are essential for the exchange of macromolecules across the nuclear envelope. A recent genetic screen in our laboratory identified a partial loss-of-function mutation in Arabidopsis MOS7/Nup88 that causes defects in basal immunity, Resistance (R) protein-mediated defense and systemic acquired resistance. In Drosophila and mammalian cells, exportin-mediated nuclear export of activated Rel/NFκB transcription factors is enhanced in nup88 mutants resulting in immune response failure. Consistent with Nup88 promoting nuclear retention of NFκB, our functional analyses revealed that MOS7/Nup88 is required for appropriate nuclear accumulation of the autoactivated R protein snc1, as well as the key immune regulators EDS1 and NPR1. These results suggest that controlling the nuclear concentrations of specific immune regulators is fundamental for defining defense outputs.

A novel Minimalist Cell-Free MHC Class II Antigen Processing System Identifies Immunodominant Epitopes

PubMed Central

Hartman, Isamu Z.; Kim, AeRyon; Cotter, Robert J.; Walter, Kimberly; Dalai, Sarat K.; Boronina, Tatiana; Griffith, Wendell; Schwenk, Robert; Lanar, David E.; Krzych, Urszula; Cole, Robert N.; Sadegh-Nasseri, Scheherazade

2010-01-01

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4+ T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: MHC class II, cathepsins, and HLA-DM. Our minimalist system successfully identified the physiologically selected immunodominant epitopes of model antigens, HA1 from influenza virus (A/Texas/1/77) and type II collagen. When applied for de novo epitope identification to a malaria antigen, or HA1 from H5N1 virus (Avian Flu), the system selected a single epitope from each protein that were confirmed to be immunodominant by their capacity to activate CD4+ T cells in HLA-DR1 positive human volunteers or transgenic mice immunized with the corresponding proteins. Thus, we provide a powerful new tool for the identification of physiologically relevant helper T cell epitopes from antigens. PMID:21037588

Molecular dynamics simulations of lysozyme-lipid systems: probing the early steps of protein aggregation.

PubMed

Trusova, Valeriya M; Gorbenko, Galyna P

2017-07-10

Using the molecular dynamics simulation, the role of lipids in the lysozyme transition into the aggregation-competent conformation has been clarified. Analysis of the changes of lysozyme secondary structure upon its interactions with the model bilayer membranes composed of phosphatidylcholine and its mixtures with phosphatidylglycerol (10, 40, and 80 mol%) within the time interval of 100 ns showed that lipid-bound protein is characterized by the increased content of β-structures. Along with this, the formation of protein-lipid complexes was accompanied by the increase in the gyration radius and the decrease in RMSD of polypeptide chain. The results obtained were interpreted in terms of the partial unfolding of lysozyme molecule on the lipid matrix, with the magnitude of this effect being increased with increasing the fraction of anionic lipids. Based on the results of molecular dynamics simulation, a hypothetical model of the nucleation of lysozyme amyloid fibrils in a membrane environment was suggested.

Expression, purification and biochemical characterization of the cytoplasmic loop of PomA, a stator component of the Na+ driven flagellar motor

PubMed Central

Abe-Yoshizumi, Rei; Kobayashi, Shiori; Gohara, Mizuki; Hayashi, Kokoro; Kojima, Chojiro; Kojima, Seiji; Sudo, Yuki; Asami, Yasuo; Homma, Michio

2013-01-01

Flagellar motors embedded in bacterial membranes are molecular machines powered by specific ion flows. Each motor is composed of a stator and a rotor and the interactions of those components are believed to generate the torque. Na+ influx through the PomA/PomB stator complex of Vibrio alginolyticus is coupled to torque generation and is speculated to trigger structural changes in the cytoplasmic domain of PomA that interacts with a rotor protein in the C-ring, FliG, to drive the rotation. In this study, we tried to overproduce the cytoplasmic loop of PomA (PomA-Loop), but it was insoluble. Thus, we made a fusion protein with a small soluble tag (GB1) which allowed us to express and characterize the recombinant protein. The structure of the PomA-Loop seems to be very elongated or has a loose tertiary structure. When the PomA-Loop protein was produced in E. coli, a slight dominant effect was observed on motility. We conclude that the cytoplasmic loop alone retains a certain function. PMID:27493537

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring.

PubMed

McDonald, Nathan A; Lind, Abigail L; Smith, Sarah E; Li, Rong; Gould, Kathleen L

2017-09-15

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0-80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80-160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160-350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring

PubMed Central

McDonald, Nathan A; Lind, Abigail L; Smith, Sarah E; Li, Rong

2017-01-01

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0–80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80–160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160–350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function. PMID:28914606

Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria.

PubMed

Kim, Jin-Sik; Song, Saemee; Lee, Minho; Lee, Seunghwa; Lee, Kangseok; Ha, Nam-Chul

2016-03-01

The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

A biomimetic pH-responsive polymer directs endosomal release and intracellular delivery of an endocytosed antibody complex.

PubMed

Lackey, Chantal A; Press, Oliver W; Hoffman, Allan S; Stayton, Patrick S

2002-01-01

Poly(propylacrylic acid) (PPAAc) is a synthetic pH-responsive polymer that has been shown to disrupt cell membranes at low pH values typical of the endosome, but not at physiological pH, suggesting its use as an endosomal-releasing agent [Murthy et al. J. Controlled Release 61, 137-43]. We have constructed an antibody-targeted biotherapeutic model to investigate whether PPAAc can enhance intracellular trafficking of proteins to the cytoplasm. A ternary complex composed of a biotinylated anti-CD3 antibody, streptavidin, and biotinylated PPAAc was fluorescently labeled, and its intracellular fate was analyzed by confocal microscopy, flow cytometry, and quantitative western blotting of cell fractionates. The 64.1 anti-CD3 antibody was previously shown to direct receptor-mediated endocytosis in the Jurkat T-cell lymphoma cell line and was rapidly trafficked from the endosome to the lysosomal compartment. The antibody-streptavidin complex was also rapidly internalized to the endosomal/lysosomal compartment and retained there, as evidenced by punctate regions of fluorescence observed by confocal fluorescence microscopy. In samples containing the ternary complex of antibody, streptavidin, and PPAAc-biotin, diffuse fluorescence in the cytoplasm was observed, indicating that PPAAc enhanced translocation to the cytoplasm. This was confirmed by western blotting analysis of the isolated cytoplasm. Flow cytometry results demonstrated that neither streptavidin nor PPAAc caused nonspecific uptake of the complex, nor did they inhibit antibody-mediated endocytosis. The striking enhancement of protein delivery to the cytoplasm by complexed PPAAc suggests that this polymer could provide a new delivery agent for therapeutic, vaccine, and diagnostics development.

The mate recognition protein gene mediates reproductive isolation and speciation in the Brachionus plicatilis cryptic species complex.

PubMed

Gribble, Kristin E; Mark Welch, David B

2012-08-01

Chemically mediated prezygotic barriers to reproduction likely play an important role in speciation. In facultatively sexual monogonont rotifers from the Brachionus plicatilis cryptic species complex, mate recognition of females by males is mediated by the Mate Recognition Protein (MRP), a globular glycoprotein on the surface of females, encoded by the mmr-b gene family. In this study, we sequenced mmr-b copies from 27 isolates representing 11 phylotypes of the B. plicatilis species complex, examined the mode of evolution and selection of mmr-b, and determined the relationship between mmr-b genetic distance and mate recognition among isolates. Isolates of the B. plicatilis species complex have 1-4 copies of mmr-b, each composed of 2-9 nearly identical tandem repeats. The repeats within a gene copy are generally more similar than are gene copies among phylotypes, suggesting concerted evolution. Compared to housekeeping genes from the same isolates, mmr-b has accumulated only half as many synonymous differences but twice as many non-synonymous differences. Most of the amino acid differences between repeats appear to occur on the outer face of the protein, and these often result in changes in predicted patterns of phosphorylation. However, we found no evidence of positive selection driving these differences. Isolates with the most divergent copies were unable to mate with other isolates and rarely self-crossed. Overall the degree of mate recognition was significantly correlated with the genetic distance of mmr-b. Discrimination of compatible mates in the B. plicatilis species complex is determined by proteins encoded by closely related copies of a single gene, mmr-b. While concerted evolution of the tandem repeats in mmr-b may function to maintain identity, it can also lead to the rapid spread of a mutation through all copies in the genome and thus to reproductive isolation. The mmr-b gene is evolving rapidly, and novel alleles may be maintained and increase in frequency via asexual reproduction. Our analyses indicate that mate recognition, controlled by MMR-B, may drive reproductive isolation and allow saltational sympatric speciation within the B. plicatilis cryptic species complex, and that this process may be largely neutral.

Physical principles of filamentous protein self-assembly kinetics

NASA Astrophysics Data System (ADS)

Michaels, Thomas C. T.; Liu, Lucie X.; Meisl, Georg; Knowles, Tuomas P. J.

2017-04-01

The polymerization of proteins and peptides into filamentous supramolecular structures is an elementary form of self-organization of key importance to the functioning biological systems, as in the case of actin biofilaments that compose the cellular cytoskeleton. Aberrant filamentous protein self-assembly, however, is associated with undesired effects and severe clinical disorders, such as Alzheimer’s and Parkinson’s diseases, which, at the molecular level, are associated with the formation of certain forms of filamentous protein aggregates known as amyloids. Moreover, due to their unique physicochemical properties, protein filaments are finding extensive applications as biomaterials for nanotechnology. With all these different factors at play, the field of filamentous protein self-assembly has experienced tremendous activity in recent years. A key question in this area has been to elucidate the microscopic mechanisms through which filamentous aggregates emerge from dispersed proteins with the goal of uncovering the underlying physical principles. With the latest developments in the mathematical modeling of protein aggregation kinetics as well as the improvement of the available experimental techniques it is now possible to tackle many of these complex systems and carry out detailed analyses of the underlying microscopic steps involved in protein filament formation. In this paper, we review some classical and modern kinetic theories of protein filament formation, highlighting their use as a general strategy for quantifying the molecular-level mechanisms and transition states involved in these processes.

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family.

PubMed

Mithöfer, A; Fliegmann, J; Neuhaus-Url, G; Schwarz, H; Ebel, J

2000-08-01

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.

The 193-Kd Vault Protein, Vparp, Is a Novel Poly(Adp-Ribose) Polymerase

PubMed Central

Kickhoefer, Valerie A.; Siva, Amara C.; Kedersha, Nancy L.; Inman, Elisabeth M.; Ruland, Cristina; Streuli, Michel; Rome, Leonard H.

1999-01-01

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of ∼350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle. PMID:10477748

A knowledge-based decision support system in bioinformatics: an application to protein complex extraction

PubMed Central

2013-01-01

Background We introduce a Knowledge-based Decision Support System (KDSS) in order to face the Protein Complex Extraction issue. Using a Knowledge Base (KB) coding the expertise about the proposed scenario, our KDSS is able to suggest both strategies and tools, according to the features of input dataset. Our system provides a navigable workflow for the current experiment and furthermore it offers support in the configuration and running of every processing component of that workflow. This last feature makes our system a crossover between classical DSS and Workflow Management Systems. Results We briefly present the KDSS' architecture and basic concepts used in the design of the knowledge base and the reasoning component. The system is then tested using a subset of Saccharomyces cerevisiae Protein-Protein interaction dataset. We used this subset because it has been well studied in literature by several research groups in the field of complex extraction: in this way we could easily compare the results obtained through our KDSS with theirs. Our system suggests both a preprocessing and a clustering strategy, and for each of them it proposes and eventually runs suited algorithms. Our system's final results are then composed of a workflow of tasks, that can be reused for other experiments, and the specific numerical results for that particular trial. Conclusions The proposed approach, using the KDSS' knowledge base, provides a novel workflow that gives the best results with regard to the other workflows produced by the system. This workflow and its numeric results have been compared with other approaches about PPI network analysis found in literature, offering similar results. PMID:23368995

Structural Basis of Rap Phosphatase Inhibition by Phr Peptides

PubMed Central

Gallego del Sol, Francisca; Marina, Alberto

2013-01-01

Two-component systems, composed of a sensor histidine kinase and an effector response regulator (RR), are the main signal transduction devices in bacteria. In Bacillus, the Rap protein family modulates complex signaling processes mediated by two-component systems, such as competence, sporulation, or biofilm formation, by inhibiting the RR components involved in these pathways. Despite the high degree of sequence homology, Rap proteins exert their activity by two completely different mechanisms of action: inducing RR dephosphorylation or blocking RR binding to its target promoter. However the regulatory mechanism involving Rap proteins is even more complex since Rap activity is antagonized by specific signaling peptides (Phr) through a mechanism that remains unknown at the molecular level. Using X-ray analyses, we determined the structure of RapF, the anti-activator of competence RR ComA, alone and in complex with its regulatory peptide PhrF. The structural and functional data presented herein reveal that peptide PhrF blocks the RapF-ComA interaction through an allosteric mechanism. PhrF accommodates in the C-terminal tetratricopeptide repeat domain of RapF by inducing its constriction, a conformational change propagated by a pronounced rotation to the N-terminal ComA-binding domain. This movement partially disrupts the ComA binding site by triggering the ComA disassociation, whose interaction with RapF is also sterically impaired in the PhrF-induced conformation of RapF. Sequence analyses of the Rap proteins, guided by the RapF-PhrF structure, unveil the molecular basis of Phr recognition and discrimination, allowing us to relax the Phr specificity of RapF by a single residue change. PMID:23526880

Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V₁-ATPase proton pump in naturally occurring urothelial tumors of the urinary bladder of cattle.

PubMed

Roperto, Sante; Russo, Valeria; Borzacchiello, Giuseppe; Urraro, Chiara; Lucà, Roberta; Esposito, Iolanda; Riccardi, Marita Georgia; Raso, Cinzia; Gaspari, Marco; Ceccarelli, Dora Maria; Galasso, Rocco; Roperto, Franco

2014-01-01

Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor β receptor. PDGFβR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as "V₁-ATPase subunit D", a component of the central stalk of the V₁-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. For the first time, a tri-component complex composed of E5/PDGFβR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V₀-ATPase sector. We suggest that the E5/PDGFβR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation.

PubMed

Shu, Qin; Krezel, Andrzej M; Cusumano, Zachary T; Pinkner, Jerome S; Klein, Roger; Hultgren, Scott J; Frieden, Carl

2016-06-28

Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG. The CsgG-CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel β-sheet with flexible N and C termini. The structure of CsgE fits well into the cryoelectron microscopy density map of the CsgG-CsgE complex. We highlight a striking feature of the electrostatic potential surface in CsgE structure and present an assembly model of the CsgG-CsgE complex. We suggest a structural mechanism of the interaction between CsgE and CsgA. Understanding curli formation can provide the information necessary to develop treatments and therapeutic agents for biofilm-related infections and may benefit the prevention and treatment of amyloid diseases. CsgE could establish a paradigm for the regulation of amyloidogenesis because of its unique role in curli formation.

Meiosis-Specific Cohesin Component, Stag3 Is Essential for Maintaining Centromere Chromatid Cohesion, and Required for DNA Repair and Synapsis between Homologous Chromosomes

PubMed Central

Hopkins, Jessica; Bedigian, Rick; Oka, Kazuhiro; Overbeek, Paul; Murray, Steve; Jordan, Philip W.

2014-01-01

Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis. PMID:24992337

Meiosis-specific cohesin component, Stag3 is essential for maintaining centromere chromatid cohesion, and required for DNA repair and synapsis between homologous chromosomes.

PubMed

Hopkins, Jessica; Hwang, Grace; Jacob, Justin; Sapp, Nicklas; Bedigian, Rick; Oka, Kazuhiro; Overbeek, Paul; Murray, Steve; Jordan, Philip W

2014-07-01

Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis.

Predicting permanent and transient protein-protein interfaces.

PubMed

La, David; Kong, Misun; Hoffman, William; Choi, Youn Im; Kihara, Daisuke

2013-05-01

Protein-protein interactions (PPIs) are involved in diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, whereas the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of PPIs. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces (PBIs) in protein surfaces. Without knowledge of the interacting partner, the method uses a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method, BindML+, performs very well in protein interface classification. A very high area under the curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient PBIs. Copyright © 2013 Wiley Periodicals, Inc.

Detergent-Induced Stabilization and Improved 3D Map of the Human Heteromeric Amino Acid Transporter 4F2hc-LAT2

PubMed Central

Harder, Daniel; Stauffer, Mirko; Jeckelmann, Jean-Marc; Brühlmann, Béla; Rosell, Albert; Ilgü, Hüseyin; Kovar, Karin; Palacín, Manuel; Fotiadis, Dimitrios

2014-01-01

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level. PMID:25299125

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

PubMed

Meury, Marcel; Costa, Meritxell; Harder, Daniel; Stauffer, Mirko; Jeckelmann, Jean-Marc; Brühlmann, Béla; Rosell, Albert; Ilgü, Hüseyin; Kovar, Karin; Palacín, Manuel; Fotiadis, Dimitrios

2014-01-01

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

Whirlin Replacement Restores the Formation of the USH2 Protein Complex in Whirlin Knockout Photoreceptors

PubMed Central

Zou, Junhuang; Luo, Ling; Shen, Zuolian; Chiodo, Vince A.; Ambati, Balamurali K.; Hauswirth, William W.

2011-01-01

Purpose. Whirlin is the causative gene for Usher syndrome type IID (USH2D), a condition manifested as both retinitis pigmentosa and congenital deafness. Mutations in this gene cause disruption of the USH2 protein complex composed of USH2A and VLGR1 at the periciliary membrane complex (PMC) in photoreceptors. In this study, the adeno-associated virus (AAV)-mediated whirlin replacement was evaluated as a treatment option. Methods. Murine whirlin cDNA driven by the human rhodopsin kinase promoter (hRK) was packaged as an AAV2/5 vector and delivered into the whirlin knockout retina through subretinal injection. The efficiency, efficacy, and safety of this treatment were examined using immunofluorescent staining, confocal imaging, immunoelectron microscopy, Western blot analysis, histologic analysis, and electroretinogram. Results. The AAV-mediated whirlin expression started at two weeks, reached its maximum level at 10 weeks, and lasted up to six months post injection. The transgenic whirlin product had a molecular size and an expression level comparable to the wild-type. It was distributed at the PMC in both rod and cone photoreceptors from the central to peripheral retina. Importantly, the transgenic whirlin restored the cellular localization and expression level of both USH2A and VLGR1 and did not cause defects in the retinal histology and function in the whirlin knockout mouse. Conclusions. Whirlin transgene recruits USH2A and VLGR1 to the PMC and is sufficient for the formation of the USH2 protein complex in photoreceptors. The combined hRK and AAV gene delivery system could be an effective gene therapy approach to treat retinal degeneration in USH2D patients. PMID:21212183

γ-secretase composed of PS1/Pen2/Aph1a can cleave Notch and APP in the absence of Nicastrin

PubMed Central

Zhao, Guojun; Liu, Zhenyi; Ilagan, Ma. Xenia G.; Kopan, Raphael

2010-01-01

γ-secretase is a multiprotein intramembrane-cleaving protease with a growing list of protein substrates including the Notch receptors and the amyloid precursor protein. The four components of γ-secretase complex - presenilin (PS), nicastrin (NCT), Pen2, and Aph1 - are all thought to be essential for activity. The catalytic domain resides within PS proteins; NCT has been suggested to be critical for substrate recognition; the contributions of Pen2 and Aph1 remain unclear. The role of NCT has been challenged recently by the observation that a critical residue (E332) in NCT, thought to be essential for γ-secretase activity, is instead involved in complex maturation. Here we report that NCT is dispensable for γ-secretase activity. NCT-independent γ-secretase activity can be detected in two independent NCT-deficient MEF lines, and blocked by the γ-secretase inhibitors DAPT and L-685,458. This catalytic activity requires prior ectodomain shedding of the substrate, and can cleave ligand-activated endogenous Notch receptors, indicating presence at the plasma membrane. siRNA knockdown experiments demonstrated that NCT-independent γ-secretase activity requires the presence of PS1, Pen2 and Aph1a but can tolerate knockdown of PS2 or Aph1b. We conclude that a PS1/Pen2/Aph1a trimeric complex is an active enzyme, displaying similar biochemical properties to those of γ-secretase and roughly 50% of its activity when normalized to PS1 NTF levels. This PS1/Pen2/Aph1a complex, however, is highly unstable. Thus, NCT acts to stabilize γ-secretase, but is not required for substrate recognition. PMID:20130175

Growth performance and feed conversion efficiency of three edible mealworm species (Coleoptera: Tenebrionidae) on diets composed of organic by-products.

PubMed

van Broekhoven, Sarah; Oonincx, Dennis G A B; van Huis, Arnold; van Loon, Joop J A

2015-02-01

Insects receive increasing attention as an alternative protein-rich food source for humans. Producing edible insects on diets composed of organic by-products could increase sustainability. In addition, insect growth rate and body composition, and hence nutritional quality, can be altered by diet. Three edible mealworm species Tenebrio molitor L., Zophobas atratus Fab. and Alphitobius diaperinus Panzer were grown on diets composed of organic by-products originating from beer brewing, bread/cookie baking, potato processing and bioethanol production. Experimental diets differed with respect to protein and starch content. Larval growth and survival was monitored. Moreover, effects of dietary composition on feed conversion efficiency and mealworm crude protein and fatty acid profile were assessed. Diet affected mealworm development and feed conversion efficiency such that diets high in yeast-derived protein appear favourable, compared to diets used by commercial breeders, with respect to shortening larval development time, reducing mortality and increasing weight gain. Diet also affected the chemical composition of mealworms. Larval protein content was stable on diets that differed 2-3-fold in protein content, whereas dietary fat did have an effect on larval fat content and fatty acid profile. However, larval fatty acid profile did not necessarily follow the same trend as dietary fatty acid composition. Diets that allowed for fast larval growth and low mortality in this study led to a comparable or less favourable n6/n3 fatty acid ratio compared to control diets used by commercial breeders. In conclusion, the mealworm species used in this study can be grown successfully on diets composed of organic by-products. Diet composition did not influence larval protein content, but did alter larval fat composition to a certain extent. Copyright © 2015 Elsevier Ltd. All rights reserved.

Spike-In Normalization of ChIP Data Using DNA-DIG-Antibody Complex.

PubMed

Eberle, Andrea B

2018-01-01

Chromatin immunoprecipitation (ChIP) is a widely used method to determine the occupancy of specific proteins within the genome, helping to unravel the function and activity of specific genomic regions. In ChIP experiments, normalization of the obtained data by a suitable internal reference is crucial. However, particularly when comparing differently treated samples, such a reference is difficult to identify. Here, a simple method to improve the accuracy and reliability of ChIP experiments by the help of an external reference is described. An artificial molecule, composed of a well-defined digoxigenin (DIG) labeled DNA fragment in complex with an anti-DIG antibody, is synthesized and added to each chromatin sample before immunoprecipitation. During the ChIP procedure, the DNA-DIG-antibody complex undergoes the same treatments as the chromatin and is therefore purified and quantified together with the chromatin of interest. This external reference compensates for variability during the ChIP routine and improves the similarity between replicates, thereby emphasizing the biological differences between samples.

Assembly and function of the botulinum neurotoxin progenitor complex.

PubMed

Gu, Shenyan; Jin, Rongsheng

2013-01-01

Botulinum neurotoxins (BoNTs) are among the most poisonous substances known to man, but paradoxically, BoNT-containing medicines and cosmetics have been used with great success in the clinic. Accidental BoNT poisoning mainly occurs through oral ingestion of food contaminated with Clostridium botulinum. BoNTs are naturally produced in the form of progenitor toxin complexes (PTCs), which are high molecular weight (up to ~900 kDa) multiprotein complexes composed of BoNT and several non-toxic neurotoxin-associated proteins (NAPs). NAPs protect the inherently fragile BoNTs against the hostile environment of the gastrointestinal (GI) tract and help BoNTs pass through the intestinal epithelial barrier before they are released into the general circulation. These events are essential for ingested BoNTs to gain access to motoneurons, where they inhibit neurotransmitter release and cause muscle paralysis. In this review, we discuss the structural basis for assembly of NAPs and BoNT into the PTC that protects BoNT and facilitate its delivery into the bloodstream.

Clarifying the supercomplex: the higher-order organization of the mitochondrial electron transport chain.

PubMed

Letts, James A; Sazanov, Leonid A

2017-10-05

The oxidative phosphorylation electron transport chain (OXPHOS-ETC) of the inner mitochondrial membrane is composed of five large protein complexes, named CI-CV. These complexes convert energy from the food we eat into ATP, a small molecule used to power a multitude of essential reactions throughout the cell. OXPHOS-ETC complexes are organized into supercomplexes (SCs) of defined stoichiometry: CI forms a supercomplex with CIII 2 and CIV (SC I+III 2 +IV, known as the respirasome), as well as with CIII 2 alone (SC I+III 2 ). CIII 2 forms a supercomplex with CIV (SC III 2 +IV) and CV forms dimers (CV 2 ). Recent cryo-EM studies have revealed the structures of SC I+III 2 +IV and SC I+III 2 . Furthermore, recent work has shed light on the assembly and function of the SCs. Here we review and compare these recent studies and discuss how they have advanced our understanding of mitochondrial electron transport.

Identification and Characterization of Components of a Putative Petunia S-Locus F-Box–Containing E3 Ligase Complex Involved in S-RNase–Based Self-Incompatibility[W

PubMed Central

Hua, Zhihua; Kao, Teh-hui

2006-01-01

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF2. We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S1-, S2-, and S3-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S3-RNase is not degraded to any significant extent; however, deglycosylated S3-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown. PMID:17028207

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex* ♦

PubMed Central

Nadeau, Owen W.; Lane, Laura A.; Xu, Dong; Sage, Jessica; Priddy, Timothy S.; Artigues, Antonio; Villar, Maria T.; Yang, Qing; Robinson, Carol V.; Zhang, Yang; Carlson, Gerald M.

2012-01-01

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)4 complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)2 lobes joined with D2 symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)4 complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)4 kinase core, because a β4 subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit. PMID:22969083

Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction

PubMed Central

Becker, Matthias M. M.; Lapouge, Karine; Segnitz, Bernd; Wild, Klemens; Sinning, Irmgard

2017-01-01

Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway. PMID:27899666

Adaptive evolution of centromere proteins in plants and animals.

PubMed

Talbert, Paul B; Bryson, Terri D; Henikoff, Steven

2004-01-01

Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Drosophila and Arabidopsis. Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif). Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection. CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi.

Comprehensive Characterization of AMP-activated Protein Kinase Catalytic Domain by Top-down Mass Spectrometry

PubMed Central

Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

2015-01-01

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ. C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ has noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems. PMID:26489410

Adaptive evolution of centromere proteins in plants and animals

PubMed Central

Talbert, Paul B; Bryson, Terri D; Henikoff, Steven

2004-01-01

Background Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Drosophila and Arabidopsis. Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif). Results Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection. Conclusions CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi. PMID:15345035

Effect of Periodontal Pathogens on the Metatranscriptome of a Healthy Multispecies Biofilm Model

PubMed Central

Duran-Pinedo, Ana

2012-01-01

Oral bacterial biofilms are highly complex microbial communities with up to 700 different bacterial taxa. We report here the use of metatranscriptomic analysis to study patterns of community gene expression in a multispecies biofilm model composed of species found in healthy oral biofilms (Actinomyces naeslundii, Lactobacillus casei, Streptococcus mitis, Veillonella parvula, and Fusobacterium nucleatum) and the same biofilm plus the periodontopathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The presence of the periodontopathogens altered patterns in gene expression, and data indicate that transcription of protein-encoding genes and small noncoding RNAs is stimulated. In the healthy biofilm hypothetical proteins, transporters and transcriptional regulators were upregulated while chaperones and cell division proteins were downregulated. However, when the pathogens were present, chaperones were highly upregulated, probably due to increased levels of stress. We also observed a significant upregulation of ABC transport systems and putative transposases. Changes in Clusters of Orthologous Groups functional categories as well as gene set enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that in the absence of pathogens, only sets of proteins related to transport and secondary metabolism were upregulated, while in the presence of pathogens, proteins related to growth and division as well as a large portion of transcription factors were upregulated. Finally, we identified several small noncoding RNAs whose predicted targets were genes differentially expressed in the open reading frame libraries. These results show the importance of pathogens controlling gene expression of a healthy oral community and the usefulness of metatranscriptomic techniques to study gene expression profiles in complex microbial community models. PMID:22328675

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex.

PubMed

Ma, Jiong; Kelich, Joseph M; Junod, Samuel L; Yang, Weidong

2017-04-01

The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes. © 2017. Published by The Company of Biologists Ltd.

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex

PubMed Central

Ma, Jiong; Kelich, Joseph M.; Junod, Samuel L.

2017-01-01

ABSTRACT The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes. PMID:28202688

In vitro synthesis of the iron–molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins

PubMed Central

Curatti, Leonardo; Hernandez, Jose A.; Igarashi, Robert Y.; Soboh, Basem; Zhao, Dehua; Rubio, Luis M.

2007-01-01

Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron–molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe2+, S2−, MoO42−, and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe2+, S2−, MoO42−, R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed. PMID:17978192

Nano-scale characterization of the dynamics of the chloroplast Toc translocon.

PubMed

Reddick, L Evan; Chotewutmontri, Prakitchai; Crenshaw, Will; Dave, Ashita; Vaughn, Michael; Bruce, Barry D

2008-01-01

Translocons are macromolecular nano-scale machines that facilitate the selective translocation of proteins across membranes. Although common in function, different translocons have evolved diverse molecular mechanisms for protein translocation. Subcellular organelles of endosymbiotic origin such as the chloroplast and mitochondria had to evolve/acquire translocons capable of importing proteins whose genes were transferred to the host genome. These gene products are expressed on cytosolic ribosomes as precursor proteins and targeted back to the organelle by an N-terminal extension called the transit peptide or presequence. In chloroplasts the transit peptide is specifically recognized by the Translocon of the Outer Chloroplast membrane (Toc) which is composed of receptor GTPases that potentially function as gate-like switches, where GTP binding and hydrolysis somehow facilitate preprotein binding and translocation. Compared to other translocons, the dynamics of the Toc translocon are probably more complex and certainly less understood. We have developed biochemical/biophysical, imaging, and computational techniques to probe the dynamics of the Toc translocon at the nanoscale. In this chapter we provide detailed protocols for kinetic and binding analysis of precursor interactions in organeller, measurement of the activity and nucleotide binding of the Toc GTPases, native electrophoretic analysis of the assembly/organization of the Toc complex, visualization of the distribution and mobility of Toc apparatus on the surface of chloroplasts, and conclude with the identification and molecular modeling Toc75 POTRA domains. With these new methodologies we discuss future directions of the field.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1).

PubMed

Fonseca, Bruno D; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M; Diao, Ilo T; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L; Hernández, Greco; Alain, Tommy; Damgaard, Christian K

2015-06-26

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The vaccinia virus I3L gene product is localized to a complex endoplasmic reticulum-associated structure that contains the viral parental DNA.

PubMed

Welsch, Sonja; Doglio, Laura; Schleich, Sibylle; Krijnse Locker, Jacomine

2003-05-01

The vaccinia virus (VV) I3L gene product is a single-stranded DNA-binding protein made early in infection that localizes to the cytoplasmic sites of viral DNA replication (S. C. Rochester and P. Traktman, J. Virol. 72:2917-2926, 1998). Surprisingly, when replication was blocked, the protein localized to distinct cytoplasmic spots (A. Domi and G. Beaud, J. Gen. Virol. 81:1231-1235, 2000). Here these I3L-positive spots were characterized in more detail. By using an anti-I3L peptide antibody we confirmed that the protein localized to the cytoplasmic sites of viral DNA replication by both immunofluorescence and electron microscopy (EM). Before replication had started or when replication was inhibited with hydroxyurea or cytosine arabinoside, I3L localized to distinct cytoplasmic punctate structures of homogeneous size. We show that these structures are not incoming cores or cytoplasmic sites of VV early mRNA accumulation. Instead, morphological and quantitative data indicate that they are specialized sites where the parental DNA accumulates after its release from incoming viral cores. By EM, these sites appeared as complex, electron-dense structures that were intimately associated with the cellular endoplasmic reticulum (ER). By double labeling of cryosections we show that they contain DNA and a viral early protein, the gene product of E8R. Since E8R is a membrane protein that is able to bind to DNA, the localization of this protein to the I3L puncta suggests that they are composed of membranes. The results are discussed in relation to our previous data showing that the process of viral DNA replication also occurs in close association with the ER.

The protein domains of the Dictyostelium microprocessor that are required for correct subcellular localization and for microRNA maturation.

PubMed

Kruse, Janis; Meier, Doreen; Zenk, Fides; Rehders, Maren; Nellen, Wolfgang; Hammann, Christian

2016-10-02

The maturation pathways of microRNAs (miRNAs) have been delineated for plants and several animals, belonging to the evolutionary supergroups of Archaeplastida and Opisthokonta, respectively. Recently, we reported the discovery of the microprocessor complex in Dictyostelium discoideum of the Amoebozoa supergroup. The complex is composed of the Dicer DrnB and the dsRBD (double-stranded RNA binding domain) containing protein RbdB. Both proteins localize at nucleoli, where they physically interact, and both are required for miRNA maturation. Here we show that the miRNA phenotype of a ΔdrnB gene deletion strain can be rescued by ectopic expression of a series of DrnB GFP fusion proteins, which consistently showed punctate perinucleolar localization in fluorescence microscopy. These punctate foci appear surprisingly stable, as they persist both disintegration of nucleoli and degradation of cellular nucleic acids. We observed that DrnB expression levels influence the number of microprocessor foci and alter RbdB accumulation. An investigation of DrnB variants revealed that its newly identified nuclear localization signal is necessary, but not sufficient for the perinucleolar localization. Biogenesis of miRNAs, which are RNA Pol II transcripts, is correlated with that localization. Besides its bidentate RNase III domains, DrnB contains only a dsRBD, which surprisingly is dispensable for miRNA maturation. This dsRBD can, however, functionally replace the homologous domain in RbdB. Based on the unique setup of the Dictyostelium microprocessor with a subcellular localization similar to plants, but a protein domain composition similar to animals, we propose a model for the evolutionary origin of RNase III proteins acting in miRNA maturation.

The Ever-Evolving Concept of the Gene: The Use of RNA/Protein Experimental Techniques to Understand Genome Functions

PubMed Central

Cipriano, Andrea; Ballarino, Monica

2018-01-01

The completion of the human genome sequence together with advances in sequencing technologies have shifted the paradigm of the genome, as composed of discrete and hereditable coding entities, and have shown the abundance of functional noncoding DNA. This part of the genome, previously dismissed as “junk” DNA, increases proportionally with organismal complexity and contributes to gene regulation beyond the boundaries of known protein-coding genes. Different classes of functionally relevant nonprotein-coding RNAs are transcribed from noncoding DNA sequences. Among them are the long noncoding RNAs (lncRNAs), which are thought to participate in the basal regulation of protein-coding genes at both transcriptional and post-transcriptional levels. Although knowledge of this field is still limited, the ability of lncRNAs to localize in different cellular compartments, to fold into specific secondary structures and to interact with different molecules (RNA or proteins) endows them with multiple regulatory mechanisms. It is becoming evident that lncRNAs may play a crucial role in most biological processes such as the control of development, differentiation and cell growth. This review places the evolution of the concept of the gene in its historical context, from Darwin's hypothetical mechanism of heredity to the post-genomic era. We discuss how the original idea of protein-coding genes as unique determinants of phenotypic traits has been reconsidered in light of the existence of noncoding RNAs. We summarize the technological developments which have been made in the genome-wide identification and study of lncRNAs and emphasize the methodologies that have aided our understanding of the complexity of lncRNA-protein interactions in recent years. PMID:29560353

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1)*

PubMed Central

Fonseca, Bruno D.; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E.; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M.; Diao, Ilo T.; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M.; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L.; Hernández, Greco; Alain, Tommy; Damgaard, Christian K.

2015-01-01

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5′TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. PMID:25940091

Fueling the Flames: Mammalian Programmed Necrosis in Inflammatory Diseases

PubMed Central

Chan, Francis Ka-Ming

2012-01-01

Programmed necrosis or necroptosis is an inflammatory form of cell death driven by TNF-like death cytokines, toll-like receptors, and antigen receptors. Unlike necrosis induced by physical trauma, a dedicated pathway is involved in programmed necrosis. In particular, a kinase complex composed of the receptor interacting protein kinase 1 (RIPK1) and RIPK3 is a central step in necrotic cell death. Assembly and activation of this RIPK1–RIPK3 “necrosome” is critically controlled by protein ubiquitination, phosphorylation, and caspase-mediated cleavage events. The molecular signals cumulate in formation of intracellular vacuoles, organelle swelling, internal membrane leakage, and eventually plasma membrane rupture. These morphological changes can result in spillage of intracellular adjuvants to promote inflammation and further exacerbate tissue injury. Because of the inflammatory nature of necrosis, it is an attractive pathway for therapeutic intervention in acute inflammatory diseases. PMID:23125016

Analyte-Triggered DNA-Probe Release from a Triplex Molecular Beacon for Nanopore Sensing.

PubMed

Guo, Bingyuan; Sheng, Yingying; Zhou, Ke; Liu, Quansheng; Liu, Lei; Wu, Hai-Chen

2018-03-26

A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex-forming molecular beacon and a stem-forming DNA component that is modified with a host-guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α-hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point-of-care diagnostics with a portable nanopore kit in the future. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Role of Activator Protein-1 (AP-1) Family Members in CD30-Positive Lymphomas

PubMed Central

Garces de los Fayos Alonso, Ines; Lagger, Sabine; Merkel, Olaf; Kenner, Lukas

2018-01-01

The Activator Protein-1 (AP-1) transcription factor (TF) family, composed of a variety of members including c-JUN, c-FOS and ATF, is involved in mediating many biological processes such as proliferation, differentiation and cell death. Since their discovery, the role of AP-1 TFs in cancer development has been extensively analysed. Multiple in vitro and in vivo studies have highlighted the complexity of these TFs, mainly due to their cell-type specific homo- or hetero-dimerization resulting in diverse transcriptional response profiles. However, as a result of the increasing knowledge of the role of AP-1 TFs in disease, these TFs are being recognized as promising therapeutic targets for various malignancies. In this review, we focus on the impact of deregulated expression of AP-1 TFs in CD30-positive lymphomas including Classical Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma. PMID:29597249

A Role for Small Antibody Fragments to Bind and Neutralize HIV | Center for Cancer Research

Cancer.gov

The surface of the Human Immunodeficiency Virus (HIV) is studded with numerous copies of the glycoprotein Env. Each Env spike is composed of three copies of the proteins gp41, which sits in the viral membrane, and gp120, which rests on top of each gp41 molecule. Env is essential for HIV-mediated infection because the binding of gp120 to the T cell surface receptor CD4 initiates a conformational change in Env exposing the fusion peptide, which inserts into the T cell membrane and helps fuse the T cell and virus together. This makes Env an attractive target for designing therapeutic inhibitory antibodies. However, the complexities of the HIV surface proteins and the tight association of the virus and T cell during infection have hampered the identification of full-length antibodies with effective HIV neutralizing activity.

Pleiotropic effects of mutations involved in the regulation of Escherichia coli K-12 alkaline phosphatase.

PubMed

Morris, H; Schlesinger, M J; Bracha, M; Yagil, E

1974-08-01

Induction of alkaline phosphatase in wild-type Escherichia coli K-12 leads to the appearance of three new proteins in addition to alkaline phosphatase in the periplasmic space of the bacteria. These proteins are detected in autoradiograms of sodium dodecyl sulfate-acrylamide gel electropherograms of extracts from cells labeled with [(35)S]methionine. Studies with constitutive mutants defective in the three genes phoS, phoT, and phoR that have been shown to regulate alkaline phosphatase synthesis indicate that the three periplasmic proteins are coregulated with alkaline phosphatase. A mutant that has a deletion in the alkaline phosphatase structural gene phoA produces the three proteins, but a newly discovered mutant phoB that has a defect in the expression of alkaline phosphatase fails to produce the three proteins. phoB mutants are shown here to be unable to make detectable amounts of alkaline phosphatase polypeptides, as measured by immunoprecipitins or acrylamide gel electropherograms. On the basis of these results we suggest a new model for the regulation of alkaline phosphatase biosynthesis. In this model, a ternary complex composed of phoB(+) and phoR(+) gene products and an internal metabolite functions as a positive control element to regulate the transcription of several cistrons coding for periplasmic proteins.

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

AhYoung, Andrew P.; Lu, Brian; Cascio, Duilio

Membrane contact sites between organelles serve as molecular hubs for the exchange of metabolites and signals. In yeast, the Endoplasmic Reticulum – Mitochondrion Encounter Structure (ERMES) tethers these two organelles likely to facilitate the non-vesicular exchange of essential phospholipids. Present in Fungi and Amoebas but not in Metazoans, ERMES is composed of five distinct subunits; among those, Mdm12, Mmm1 and Mdm34 each contain an SMP domain functioning as a lipid transfer module. We previously showed that the SMP domains of Mdm12 and Mmm1 form a hetero-tetramer. Here we describe our strategy to diversify the number of Mdm12/Mmm1 complexes suited formore » structural studies. We use sequence analysis of orthologues combined to protein engineering of disordered regions to guide the design of protein constructs and expand the repertoire of Mdm12/Mmm1 complexes more likely to crystallize. Using this combinatorial approach we report crystals of Mdm12/Mmm1 ERMES complexes currently diffracting to 4.5 Å resolution and a new structure of Mdm12 solved at 4.1 Å resolution. Our structure reveals a monomeric form of Mdm12 with a conformationally dynamic N-terminal β-strand; it differs from a previously reported homodimeric structure where the N-terminal β strands where swapped to promote dimerization. Based on our electron microscopy data, we propose a refined pseudo-atomic model of the Mdm12/Mmm1 complex that agrees with our crystallographic and small-angle X-ray scattering (SAXS) solution data.« less

Reversal of an Epigenetic Switch Governing Cell Chaining in Bacillus subtilis by Protein Instability

PubMed Central

Chai, Yunrong; Kolter, Roberto; Losick, Richard

2010-01-01

Bacillus subtilis forms long chains of cells during growth and biofilm formation. Cell separation is mediated by autolysins, whose genes are under the negative control of a heteromeric complex composed of the proteins SinR and SlrR. Formation of the SinR•SlrR complex is governed by a self-reinforcing, double-negative feedback loop in which SinR represses the gene for SlrR and SlrR, by forming the SinR•SlrR complex, titrates SinR and prevents it from repressing slrR. The loop is a bistable switch and exists in a SlrRLOW state in which autolysin genes are on, and a SlrRHIGH state in which autolysin genes are repressed by SinR•SlrR. Cells in the SlrRLOW state are driven into the SlrRHIGH state by SinI, an antirepressor that binds to and inhibits SinR. However, the mechanism by which cells in the SlrRHIGH state revert back to the SlrRLOW state is unknown. We report that SlrR is proteolytically unstable and present evidence that self-cleavage via a LexA-like autopeptidase and ClpC contribute to its degradation. Cells producing a self-cleavage-resistant mutant of SlrR exhibited more persistent chaining during growth and yielded biofilms with enhanced structural complexity. We propose that degradation of SlrR allows cells to switch from the SlrRHIGH to the SlrRLOW state. PMID:20923420

Chaperoning 5S RNA assembly.

PubMed

Madru, Clément; Lebaron, Simon; Blaud, Magali; Delbos, Lila; Pipoli, Juliana; Pasmant, Eric; Réty, Stéphane; Leulliot, Nicolas

2015-07-01

In eukaryotes, three of the four ribosomal RNAs (rRNAs)—the 5.8S, 18S, and 25S/28S rRNAs—are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit. © 2015 Madru et al.; Published by Cold Spring Harbor Laboratory Press.

Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes

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

Lafleur, Michel, E-mail: michel.lafleur@umontreal.ca; Courtemanche, Lesley; Karlsson, Goeran

Research highlights: {yields} Binder-of-sperm protein 1 (BSP1) modifies the morphology of lipidic vesicles inducing bead necklace-like and thread-like structures. {yields} In the presence of multilamellar liposomes, BSP1 leads to the formation of long nanotubes. {yields} The insertion of BSP1 in the external lipid leaflet of membranes induces local changes in bilayer curvature. -- Abstract: Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed withmore » phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process.« less

Caveolae, caveolin-1 and cavin-1: Emerging roles in pulmonary hypertension.

PubMed

Chettimada, Sukrutha; Yang, Jincheng; Moon, Hyung-Geun; Jin, Yang

2015-07-28

Caveolae are flask-shaped invaginations of cell membrane that play a significant structural and functional role. Caveolae harbor a variety of signaling molecules and serve to receive, concentrate and transmit extracellular signals across the membrane. Caveolins are the main structural proteins residing in the caveolae. Caveolins and another category of newly identified caveolae regulatory proteins, named cavins, are not only responsible for caveolae formation, but also interact with signaling complexes in the caveolae and regulate transmission of signals across the membrane. In the lung, two of the three caveolin isoforms, i.e ., cav-1 and -2, are expressed ubiquitously. Cavin protein family is composed of four proteins, named cavin-1 (or PTRF for polymerase Ⅰ and transcript release factor), cavin-2 (or SDPR for serum deprivation protein response), cavin-3 (or SRBC for sdr-related gene product that binds to-c-kinase) and cavin-4 (or MURC for muscle restricted coiled-coiled protein or cavin-4). All the caveolin and cavin proteins are essential regulators for caveolae dynamics. Recently, emerging evidence suggest that caveolae and its associated proteins play crucial roles in development and progression of pulmonary hypertension. The focus of this review is to outline and discuss the contrast in alteration of cav-1 (cav-1),-2 and cavin-1 (PTRF) expression and downstream signaling mechanisms between human and experimental models of pulmonary hypertension.

Structure and Biochemical Activities of Escherichia coli MgsA

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

Page, Asher N.; George, Nicholas P.; Marceau, Aimee H.

2012-02-27

Bacterial 'maintenance of genome stability protein A' (MgsA) and related eukaryotic enzymes play important roles in cellular responses to stalled DNA replication processes. Sequence information identifies MgsA enzymes as members of the clamp loader clade of AAA{sup +} proteins, but structural information defining the family has been limited. Here, the x-ray crystal structure of Escherichia coli MgsA is described, revealing a homotetrameric arrangement for the protein that distinguishes it from other clamp loader clade AAA{sup +} proteins. Each MgsA protomer is composed of three elements as follows: ATP-binding and helical lid domains (conserved among AAA{sup +} proteins) and a tetramerizationmore » domain. Although the tetramerization domains bury the greatest amount of surface area in the MgsA oligomer, each of the domains participates in oligomerization to form a highly intertwined quaternary structure. Phosphate is bound at each AAA{sup +} ATP-binding site, but the active sites do not appear to be in a catalytically competent conformation due to displacement of Arg finger residues. E. coli MgsA is also shown to form a complex with the single-stranded DNA-binding protein through co-purification and biochemical studies. MgsA DNA-dependent ATPase activity is inhibited by single-stranded DNA-binding protein. Together, these structural and biochemical observations provide insights into the mechanisms of MgsA family AAA{sup +} proteins.« less

Electrostatic Architecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein Illustrates a Carboxyl-Carboxylate pH Sensor.

PubMed

Cook, Jonathan D; Soto-Montoya, Hazel; Korpela, Markus K; Lee, Jeffrey E

2015-07-24

Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and Biochemical Activities of Escherichia coli MgsA*♦

PubMed Central

Page, Asher N.; George, Nicholas P.; Marceau, Aimee H.; Cox, Michael M.; Keck, James L.

2011-01-01

Bacterial “maintenance of genome stability protein A” (MgsA) and related eukaryotic enzymes play important roles in cellular responses to stalled DNA replication processes. Sequence information identifies MgsA enzymes as members of the clamp loader clade of AAA+ proteins, but structural information defining the family has been limited. Here, the x-ray crystal structure of Escherichia coli MgsA is described, revealing a homotetrameric arrangement for the protein that distinguishes it from other clamp loader clade AAA+ proteins. Each MgsA protomer is composed of three elements as follows: ATP-binding and helical lid domains (conserved among AAA+ proteins) and a tetramerization domain. Although the tetramerization domains bury the greatest amount of surface area in the MgsA oligomer, each of the domains participates in oligomerization to form a highly intertwined quaternary structure. Phosphate is bound at each AAA+ ATP-binding site, but the active sites do not appear to be in a catalytically competent conformation due to displacement of Arg finger residues. E. coli MgsA is also shown to form a complex with the single-stranded DNA-binding protein through co-purification and biochemical studies. MgsA DNA-dependent ATPase activity is inhibited by single-stranded DNA-binding protein. Together, these structural and biochemical observations provide insights into the mechanisms of MgsA family AAA+ proteins. PMID:21297161

Amino Acid Interaction (INTAA) web server.

PubMed

Galgonek, Jakub; Vymetal, Jirí; Jakubec, David; Vondrášek, Jirí

2017-07-03

Large biomolecules-proteins and nucleic acids-are composed of building blocks which define their identity, properties and binding capabilities. In order to shed light on the energetic side of interactions of amino acids between themselves and with deoxyribonucleotides, we present the Amino Acid Interaction web server (http://bioinfo.uochb.cas.cz/INTAA/). INTAA offers the calculation of the residue Interaction Energy Matrix for any protein structure (deposited in Protein Data Bank or submitted by the user) and a comprehensive analysis of the interfaces in protein-DNA complexes. The Interaction Energy Matrix web application aims to identify key residues within protein structures which contribute significantly to the stability of the protein. The application provides an interactive user interface enhanced by 3D structure viewer for efficient visualization of pairwise and net interaction energies of individual amino acids, side chains and backbones. The protein-DNA interaction analysis part of the web server allows the user to view the relative abundance of various configurations of amino acid-deoxyribonucleotide pairs found at the protein-DNA interface and the interaction energies corresponding to these configurations calculated using a molecular mechanical force field. The effects of the sugar-phosphate moiety and of the dielectric properties of the solvent on the interaction energies can be studied for the various configurations. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

PubMed

Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C

2010-01-01

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

The HsiB1C1 (TssB-TssC) Complex of the Pseudomonas aeruginosa Type VI Secretion System Forms a Bacteriophage Tail Sheathlike Structure

PubMed Central

Lossi, Nadine S.; Manoli, Eleni; Förster, Andreas; Dajani, Rana; Pape, Tillmann; Freemont, Paul; Filloux, Alain

2013-01-01

Protein secretion systems in Gram-negative bacteria evolved into a variety of molecular nanomachines. They are related to cell envelope complexes, which are involved in assembly of surface appendages or transport of solutes. They are classified as types, the most recent addition being the type VI secretion system (T6SS). The T6SS displays similarities to bacteriophage tail, which drives DNA injection into bacteria. The Hcp protein is related to the T4 bacteriophage tail tube protein gp19, whereas VgrG proteins structurally resemble the gp27/gp5 puncturing device of the phage. The tube and spike of the phage are pushed through the bacterial envelope upon contraction of a tail sheath composed of gp18. In Vibrio cholerae it was proposed that VipA and VipB assemble into a tail sheathlike structure. Here we confirm these previous data by showing that HsiB1 and HsiC1 of the Pseudomonas aeruginosa H1-T6SS assemble into tubules resulting from stacking of cogwheel-like structures showing predominantly 12-fold symmetry. The internal diameter of the cogwheels is ∼100 Å, which is large enough to accommodate an Hcp tube whose external diameter has been reported to be 85 Å. The N-terminal 212 residues of HsiC1 are sufficient to form a stable complex with HsiB1, but the C terminus of HsiC1 is essential for the formation of the tubelike structure. Bioinformatics analysis suggests that HsiC1 displays similarities to gp18-like proteins in its C-terminal region. In conclusion, we provide further structural and mechanistic insights into the T6SS and show that a phage sheathlike structure is likely to be a conserved element across all T6SSs. PMID:23341461

Nanoformulation of Brain-Derived Neurotrophic Factor with Target Receptor-Triggered-Release in the Central Nervous System.

PubMed

Jiang, Yuhang; Fay, James M; Poon, Chi-Duen; Vinod, Natasha; Zhao, Yuling; Bullock, Kristin; Qin, Si; Manickam, Devika S; Yi, Xiang; Banks, William A; Kabanov, Alexander V

2018-02-07

Brain-derived neurotrophic factor (BDNF) is identified as a potent neuroprotective and neuroregenerative agent for many neurological diseases. Regrettably, its delivery to the brain is hampered by poor serum stability and rapid brain clearance. Here, a novel nanoformulation is reported composed of a bio-compatible polymer, poly(ethylene glycol)- b -poly(L-glutamic acid) (PEG-PLE), that hosts the BDNF molecule in a nanoscale complex, termed here Nano-BDNF. Upon simple mixture, Nano-BDNF spontaneously forms uniform spherical particles with a core-shell structure. Molecular dynamics simulations suggest that binding between BDNF and PEG-PLE is mediated through electrostatic coupling as well as transient hydrogen bonding. The formation of Nano-BDNF complex stabilizes BDNF and protects it from nonspecific binding with common proteins in the body fluid, while allowing it to associate with its receptors. Following intranasal administration, the nanoformulation improves BDNF delivery throughout the brain and displays a more preferable regional distribution pattern than the native protein. Furthermore, intranasally delivered Nano-BDNF results in superior neuroprotective effects in the mouse brain with lipopolysaccharides-induced inflammation, indicating promise for further evaluation of this agent for the therapy of neurologic diseases.

Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

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

Kim, Jeong Sig; Ro, Seung-Hyun; Kim, Myungjin

2015-03-30

Sestrins are stress-inducible metabolic regulators that suppress a wide range of age- and obesity-associated pathologies, many of which are due to mTORC1 overactivation. Upon various stresses, the Sestrins inhibit mTORC1 activity through an indirect mechanism that is still unclear. GATORs are recently identified protein complexes that regulate the activity of RagB, a small GTPase essential for mTORC1 activation. GATOR1 is a GTPase activating protein (GAP) for RagB whereas GATOR2 functions as an inhibitor of GATOR1. However, how the GATORs are physiologically regulated is unknown. Here we show that Sestrin2 binds to GATOR2, and liberates GATOR1 from GATOR2-mediated inhibition. Released GATOR1more » subsequently binds to and inactivates RagB, ultimately resulting in mTORC1 suppression. Consistent with this biochemical mechanism, genetic ablation of GATOR1 nullifies the mTORC1-inhibiting effect of Sestrin2 in both cell culture and Drosophila models. Collectively, we elucidate a new signaling cascade composed of Sestrin2-GATOR2-GATOR1-RagB that mediates stress-dependent suppression of mTORC1 activity.« less

Structural and functional analyses of DM43, a snake venom metalloproteinase inhibitor from Didelphis marsupialis serum.

PubMed

Neves-Ferreira, Ana G C; Perales, Jonas; Fox, Jay W; Shannon, John D; Makino, Débora L; Garratt, Richard C; Domont, Gilberto B

2002-04-12

DM43, an opossum serum protein inhibitor of snake venom metalloproteinases, has been completely sequenced, and its disulfide bond pattern has been experimentally determined. It shows homology to human alpha(1)B-glycoprotein, a plasma protein of unknown function and a member of the immunoglobulin supergene family. Size exclusion and dynamic laser light scattering data indicated that two monomers of DM43, each composed of three immunoglobulin-like domains, associated to form a homodimer in solution. Analysis of its glycan moiety showed the presence of N-acetylglucosamine, mannose, galactose, and sialic acid, most probably forming four biantennary N-linked chains. DM43 inhibited the fibrinogenolytic activities of bothrolysin and jararhagin and formed 1:1 stoichiometric stable complexes with both metalloproteinases. DM43 was ineffective against atrolysin C or A. No complex formation was detected between DM43 and jararhagin C, indicating the essential role of the metalloproteinase domain for interaction. Homology modeling based on the crystal structure of a killer cell inhibitory receptor suggested the existence of an I-type Ig fold, a hydrophobic dimerization surface and six surface loops potentially forming the metalloproteinase-binding surface on DM43.

G-quadruplexes as sensing probes.

PubMed

Ruttkay-Nedecky, Branislav; Kudr, Jiri; Nejdl, Lukas; Maskova, Darina; Kizek, Rene; Adam, Vojtech

2013-11-28

Guanine-rich sequences of DNA are able to create tetrastranded structures known as G-quadruplexes; they are formed by the stacking of planar G-quartets composed of four guanines paired by Hoogsteen hydrogen bonding. G-quadruplexes act as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplexes form a complex with anionic porphyrin hemin and exhibit peroxidase-like activity. This review focuses on overview of sensing techniques based on G-quadruplex complexes with anionic porphyrins for detection of various analytes, including metal ions such as K+, Ca2+, Ag+, Hg2+, Cu2+, Pb2+, Sr2+, organic molecules, nucleic acids, and proteins. Principles of G-quadruplex-based detection methods involve DNA conformational change caused by the presence of analyte which leads to a decrease or an increase in peroxidase activity, fluorescence, or electrochemical signal of the used probe. The advantages of various detection techniques are also discussed.

An Arabidopsis Gene Regulatory Network for Secondary Cell Wall Synthesis

PubMed Central

Taylor-Teeples, M; Lin, L; de Lucas, M; Turco, G; Toal, TW; Gaudinier, A; Young, NF; Trabucco, GM; Veling, MT; Lamothe, R; Handakumbura, PP; Xiong, G; Wang, C; Corwin, J; Tsoukalas, A; Zhang, L; Ware, D; Pauly, M; Kliebenstein, DJ; Dehesh, K; Tagkopoulos, I; Breton, G; Pruneda-Paz, JL; Ahnert, SE; Kay, SA; Hazen, SP; Brady, SM

2014-01-01

Summary The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. Here, we present a protein-DNA network between Arabidopsis transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component. PMID:25533953

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

Wang, Liwei; Yang, Jin Kuk; Kabaleeswaran, Venkataraman

The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirementmore » for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.« less

The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2

PubMed Central

Wickliffe, Katherine E.; Lorenz, Sonja; Wemmer, David E.; Kuriyan, John; Rape, Michael

2011-01-01

Ubiquitin chains of different topologies trigger distinct functional consequences, including protein degradation and reorganization of complexes. The assembly of most ubiquitin chains is promoted by E2s, yet how these enzymes achieve linkage specificity is poorly understood. We have discovered that the K11-specific Ube2S orients the donor ubiquitin through an essential non-covalent interaction that occurs in addition to the thioester bond at the E2 active site. The E2-donor ubiquitin complex transiently recognizes the acceptor ubiquitin, primarily through electrostatic interactions. The recognition of the acceptor ubiquitin surface around Lys11, but not around other lysines, generates a catalytically competent active site, which is composed of residues of both Ube2S and ubiquitin. Our studies suggest that monomeric E2s promote linkage-specific ubiquitin chain formation through substrate-assisted catalysis. PMID:21376237

Cln6 mutants associated with neuronal ceroid lipofuscinosis are degraded in a proteasome-dependent manner.

PubMed

Oresic, Kristina; Mueller, Britta; Tortorella, Domenico

2009-06-01

NCLs (neuronal ceroid lipofuscinoses), a group of inherited neurodegenerative lysosomal storage diseases that predominantly affect children, are the result of autosomal recessive mutations within one of the nine cln genes. The wild-type cln gene products are composed of membrane and soluble proteins that localize to the lysosome or the ER (endoplasmic reticulum). However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6(G123D) and Cln6(M241T)) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most probably generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knockdown of SEL1L [sel-1 suppressor of lin-12-like (Caenorhabditis elegans)], a member of an E3 ubiquitin ligase complex involved in ER protein extraction, rescued significant amounts of Cln6(G123D) and Cln6(M241T) polypeptides. The results implicate ER quality control in the instability of the Cln variants that probably contributes to the development of NCL.

LUBAC Formation Is Impaired in the Livers of Mice with MCD-Dependent Nonalcoholic Steatohepatitis.

PubMed

Matsunaga, Yasuka; Nakatsu, Yusuke; Fukushima, Toshiaki; Okubo, Hirofumi; Iwashita, Misaki; Sakoda, Hideyuki; Fujishiro, Midori; Yamamotoya, Takeshi; Kushiyama, Akifumi; Takahashi, Shin-Ichiro; Tsuchiya, Yoshihiro; Kamata, Hideaki; Tokunaga, Fuminori; Iwai, Kazuhiro; Asano, Tomoichiro

2015-01-01

Nonalcoholic steatohepatitis (NASH) is a disorder characterized by hepatic lipid accumulation followed by the inflammation-induced death of hepatocytes and fibrosis. In this process, oxidative stress contributes to the induction of several inflammatory cytokines including TNF-α andIL-1β in macrophages, while, in hepatocytes, NF-κB reportedly induces the expressions of cell survival genes for protection from apoptosis. Recently, it was reported that the new ubiquitin ligase complex termed linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on NF-κB essential modulator (NEMO) and thereby induces NF-κB pathway activation. In this study, we demonstrated the formation of LUBAC to be impaired in the livers of NASH rodent models produced by methionine and choline deficient (MCD) diet feeding, first by either gel filtration or Blue Native-PAGE, with subsequent confirmation by western blotting. The reduction of LUBAC is likely to be attributable to markedly reduced expression of SHARPIN, one of its components. Thus, impaired LUBAC formation, which would result in insufficient NF-κB activation, may be one of the molecular mechanisms underlying the enhanced apoptotic response of hepatocytes in MCD diet-induced NASH livers.

Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex.

PubMed

Kapoor, Karan; McGill, Nicole; Peterson, Cynthia B; Meyers, Harold V; Blackburn, Michael N; Baudry, Jerome

2016-03-28

The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches.

Drosophila Symplekin localizes dynamically to the histone locus body and tricellular junctions.

PubMed

Tatomer, Deirdre C; Rizzardi, Lindsay F; Curry, Kaitlin P; Witkowski, Alison M; Marzluff, William F; Duronio, Robert J

2014-01-01

The scaffolding protein Symplekin is part of multiple complexes involved in generating and modifying the 3' end of mRNAs, including cleavage-polyadenylation, histone pre-mRNA processing and cytoplasmic polyadenylation. To study these functions in vivo, we examined the localization of Symplekin during development and generated mutations of the Drosophila Symplekin gene. Mutations in Symplekin that reduce Symplekin protein levels alter the efficiency of both poly A(+) and histone mRNA 3' end formation resulting in lethality or sterility. Histone mRNA synthesis takes place at the histone locus body (HLB) and requires a complex composed of Symplekin and several polyadenylation factors that associates with the U7 snRNP. Symplekin is present in the HLB in the early embryo when Cyclin E/Cdk2 is active and histone genes are expressed and is absent from the HLB in cells that have exited the cell cycle. During oogenesis, Symplekin is preferentially localized to HLBs during S-phase in endoreduplicating follicle cells when histone mRNA is synthesized. After the completion of endoreplication, Symplekin accumulates in the cytoplasm, in addition to the nucleoplasm, and localizes to tricellular junctions of the follicle cell epithelium. This localization depends on the RNA binding protein ypsilon schachtel. CPSF-73 and a number of mRNAs are localized at this same site, suggesting that Symplekin participates in cytoplasmic polyadenylation at tricellular junctions.

Motif structure and cooperation in real-world complex networks

NASA Astrophysics Data System (ADS)

Salehi, Mostafa; Rabiee, Hamid R.; Jalili, Mahdi

2010-12-01

Networks of dynamical nodes serve as generic models for real-world systems in many branches of science ranging from mathematics to physics, technology, sociology and biology. Collective behavior of agents interacting over complex networks is important in many applications. The cooperation between selfish individuals is one of the most interesting collective phenomena. In this paper we address the interplay between the motifs’ cooperation properties and their abundance in a number of real-world networks including yeast protein-protein interaction, human brain, protein structure, email communication, dolphins’ social interaction, Zachary karate club and Net-science coauthorship networks. First, the amount of cooperativity for all possible undirected subgraphs with three to six nodes is calculated. To this end, the evolutionary dynamics of the Prisoner’s Dilemma game is considered and the cooperativity of each subgraph is calculated as the percentage of cooperating agents at the end of the simulation time. Then, the three- to six-node motifs are extracted for each network. The significance of the abundance of a motif, represented by a Z-value, is obtained by comparing them with some properly randomized versions of the original network. We found that there is always a group of motifs showing a significant inverse correlation between their cooperativity amount and Z-value, i.e. the more the Z-value the less the amount of cooperativity. This suggests that networks composed of well-structured units do not have good cooperativity properties.

Effect of RNase E deficiency on translocon protein synthesis in an RNase E-inducible strain of enterohemorrhagic Escherichia coli O157:H7.

PubMed

Lodato, Patricia B; Thuraisamy, Thujitha; Richards, Jamie; Belasco, Joel G

2017-07-06

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that assembles a type III secretion system (T3SS) on its surface. The last portion of the T3SS, called the 'translocon', is composed of a filament and a pore complex that is inserted into the membrane of intestinal epithelial cells. The genes encoding the translocon (espADB) are part of the LEE4 operon. Their expression is regulated by a complex post-transcriptional mechanism that involves the processing of LEE4 mRNA by the essential endoribonuclease RNase E. Here, we report the construction of an EHEC strain (TEA028-rne) in which RNase E can be induced by adding IPTG to the culture medium. EHEC cells deficient in RNase E displayed an abnormal morphology and slower growth, in agreement with published observations in E. coli K-12. Under those conditions, EspA and EspB were produced at higher concentrations, and protein secretion still occurred. These results indicate that RNase E negatively regulates translocon protein synthesis and demonstrate the utility of E. coli strain TEA028-rne as a tool for investigating the influence of this ribonuclease on EHEC gene expression in vitro. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evolution of the eukaryotic dynactin complex, the activator of cytoplasmic dynein

PubMed Central

2012-01-01

Background Dynactin is a large multisubunit protein complex that enhances the processivity of cytoplasmic dynein and acts as an adapter between dynein and the cargo. It is composed of eleven different polypeptides of which eight are unique to this complex, namely dynactin1 (p150Glued), dynactin2 (p50 or dynamitin), dynactin3 (p24), dynactin4 (p62), dynactin5 (p25), dynactin6 (p27), and the actin-related proteins Arp1 and Arp10 (Arp11). Results To reveal the evolution of dynactin across the eukaryotic tree the presence or absence of all dynactin subunits was determined in most of the available eukaryotic genome assemblies. Altogether, 3061 dynactin sequences from 478 organisms have been annotated. Phylogenetic trees of the various subunit sequences were used to reveal sub-family relationships and to reconstruct gene duplication events. Especially in the metazoan lineage, several of the dynactin subunits were duplicated independently in different branches. The largest subunit repertoire is found in vertebrates. Dynactin diversity in vertebrates is further increased by alternative splicing of several subunits. The most prominent example is the dynactin1 gene, which may code for up to 36 different isoforms due to three different transcription start sites and four exons that are spliced as differentially included exons. Conclusions The dynactin complex is a very ancient complex that most likely included all subunits in the last common ancestor of extant eukaryotes. The absence of dynactin in certain species coincides with that of the cytoplasmic dynein heavy chain: Organisms that do not encode cytoplasmic dynein like plants and diplomonads also do not encode the unique dynactin subunits. The conserved core of dynactin consists of dynactin1, dynactin2, dynactin4, dynactin5, Arp1, and the heterodimeric actin capping protein. The evolution of the remaining subunits dynactin3, dynactin6, and Arp10 is characterized by many branch- and species-specific gene loss events. PMID:22726940

Purification and characterization of a novel polysaccharide-peptide complex from Clinacanthus nutans Lindau leaves.

PubMed

Huang, Danmin; Li, Yunhong; Cui, Fengjie; Chen, Jun; Sun, Jiamin

2016-02-10

A novel polysaccharide-peptide complex CNP-1-2 with molecular weight of 9.17 × 10(4) Da was obtained from Clinacanthus nutans Lindau leaves by hot water extraction, ethanol precipitation, and purification with Superdex 200 and DEAE-Sepharose Fast Flow column chromatography. CNP-1-2 exhibited the highest growth inhibitory effect on human gastric cancer cells SGC-7901 with inhibition ratio of 92.34% and stimulated activation of macrophages with NO secretion level of 47.53 μmol/L among the polysaccharide fractions. CNP-1-2 comprised approximately 87.25% carbohydrate and 9.37% protein. Monosaccharide analysis suggested that CNP-1-2 was composed of L-rhamnose, l-arabinose, D-mannose, D-glucose and D-galactose with a molar ratio of 1.30:1.00:2.56:4.95:5.09. Methylation analysis, FT-IR, and (1)H NMR spectroscopy analysis revealed that CNP-1-2 might have a backbone consisting of 1,4-linked Glcp, 1,3-linked Glcp, 1,3-linked Manp, 1,4-linked Galp, 1,2,6-linked Galp and 1,2,6-linked Galp. Its side chain might be composed of 1-linked Araf, 1,6-linked Galp and 1-linked Rhap residues. AFM (atomic force micrograph) analysis revealed that CNP-1-2 had the molecular aggregation along with branched and entangled structure. Copyright © 2015 Elsevier Ltd. All rights reserved.

Immune complex-based vaccine for pig protection against parvovirus.

PubMed

Roić, B; Cajavec, S; Ergotić, N; Lipej, Z; Madić, J; Lojkić, M; Pokrić, B

2006-02-01

The insoluble immune complexes (ICs) were prepared under the conditions of double immunodiffusion in gel, using the suspension of the ultrasound treated PK-15 cell-line infected with porcine parvovirus (PPV) containing both viral particles and viral proteins, as well as pig or rabbit anti-PPV polyclonal immune sera. The immunodiffusion performed in an agarose gel allows only viral subunits with a molecular mass equal to or less than 1000 kDa, rather than the viral particles, to diffuse through the gel and reach the point where the immunoprecipitate is to be formed. The immunoprecipitation under the conditions of the diffusion ensures the optimal, i.e. equimolar ratio of both immunoprecipitating components, antibody/antigen in the IC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blot analyses showed the ICs were composed of two proteins, a protein in which molecular mass corresponded to the VP2 of the PPV and a protein with a molecular mass of the IgG. This suggests that the ICs are mainly composed of the VP2 antigen and IgG class antibodies. The potency of the IC-vaccines prepared in the form of a water-in-oil-in-water emulsion was compared with that of a commercially available, inactivated oil vaccine. The vaccination of gilts, 6 weeks before mating, with the IC containing allogeneic pig antibodies, resulted in the development of high and long-lasting anti-PPV antibody titres, similar to those generated by the licenced vaccine (P > 0.01). The content of the virus material administered by the IC was twice lower than that in the licenced vaccine. Neither systemic nor local reactions were observed in the gilts during the period of the trial with the IC vaccine. The number of viable piglets per litter varied between 9 and 12 and no signs of the PPV infection were detected. Rabbits were used as one of the alternative laboratory animal models accepted for the testing of the vaccine against the PPV. The rabbit humoral immune response generated by the IC containing the allogeneic antibodies were higher than that generated by the ICs containing the xenogeneic pig antibodies. It was similar to that generated by two-times higher content of the virus material administered by a commercially available vaccine. The IC-based vaccines belong to non-replicating, subunit vaccines, which are both ecologically convenient and the safest vaccines of all.

Expression, purification and characterization of two truncated peste des petits ruminants virus matrix proteins in Escherichia coli, and production of polyclonal antibodies against this protein.

PubMed

Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Liu, Zengshan; Wang, Zhiliang

2013-09-01

Peste des petits ruminants virus (PPRV), the etiological agent of peste des petits ruminants, is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) gene is composed of 1483 base pairs, encoding a 335 amino acids M protein with a molecular weight of approximately 38kD. We have demonstrated previously that the full-length M protein was expressed at an extremely low level or not even expressed in Escherichia coli BL21 (DE3). In this study, the M protein was split into two truncated forms to be successfully expressed in E. coli at a high level using the pET30a (+) vector, respectively, by analysis of SDS-PAGE, western blot and MALDI-TOF-MS. The optimization of culture conditions led us to perform the recombinant protein induction with 0.2mM IPTG at 28°C for 12h, whereby both proteins nevertheless were expressed in the insoluble form. Therefore, both His-tagged proteins were purified under the denaturing condition using a commercially available kit. Balb/c mice were immunized with the complex of purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by the analysis of ELISA. The specificity of the prepared polyclonal antibodies was checked by western blot and immunofluorescence, revealing them with the desirable specificity against both non-denatured and denatured M proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

The complex that inserts lipopolysaccharide into the bacterial outer membrane forms a two-protein plug-and-barrel.

PubMed

Freinkman, Elizaveta; Chng, Shu-Sin; Kahne, Daniel

2011-02-08

The cell surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS). This glycolipid is found exclusively in the outer leaflet of the asymmetric outer membrane (OM), where it forms a barrier to the entry of toxic hydrophobic molecules into the cell. LPS typically contains six fatty acyl chains and up to several hundred sugar residues. It is biosynthesized in the cytosol and must then be transported across two membranes and an aqueous intermembrane space to the cell surface. These processes are required for the viability of most Gram-negative organisms. The integral membrane β-barrel LptD and the lipoprotein LptE form an essential complex in the OM, which is necessary for LPS assembly. It is not known how this complex translocates large, amphipathic LPS molecules across the OM to the outer leaflet. Here, we show that LptE resides within the LptD β-barrel both in vitro and in vivo. LptD/E associate via an extensive interface; in one specific interaction, LptE contacts a predicted extracellular loop of LptD through the lumen of the β-barrel. Disrupting this interaction site compromises the biogenesis of LptD. This unprecedented two-protein plug-and-barrel architecture suggests how LptD/E can insert LPS from the periplasm directly into the outer leaflet of the OM to establish the asymmetry of the bilayer.

Structural basis for MTR4-ZCCHC8 interactions that stimulate the MTR4 helicase in the nuclear exosome-targeting complex.

PubMed

Puno, M Rhyan; Lima, Christopher D

2018-06-12

The nuclear exosome-targeting (NEXT) complex functions as an RNA exosome cofactor and is involved in surveillance and turnover of aberrant transcripts and noncoding RNAs. NEXT is a ternary complex composed of the RNA-binding protein RBM7, the scaffold zinc-knuckle protein ZCCHC8, and the helicase MTR4. While RNA interactions with RBM7 are known, it remains unclear how NEXT subunits collaborate to recognize and prepare substrates for degradation. Here, we show that MTR4 helicase activity is enhanced when associated with RBM7 and ZCCHC8. While uridine-rich substrates interact with RBM7 and are preferred, optimal activity is observed when substrates include a polyadenylated 3' end. We identify a bipartite interaction of ZCCHC8 with MTR4 and uncover a role for the conserved C-terminal domain of ZCCHC8 in stimulating MTR4 helicase and ATPase activities. A crystal structure reveals that the ZCCHC8 C-terminal domain binds the helicase core in a manner that is distinct from that observed for Saccharomyces cerevisiae exosome cofactors Trf4p and Air2p. Our results are consistent with a model whereby effective targeting of substrates by NEXT entails recognition of elements within the substrate and activation of MTR4 helicase activity. Copyright © 2018 the Author(s). Published by PNAS.

Integration of growth factor signals at the c-fos serum response element.

PubMed

Price, M A; Hill, C; Treisman, R

1996-04-29

A transcription factor ternary complex composed of serum response factor (SRF) and a second factor, ternary complex factor (TCF), mediates the response of the c-fos Serum Response Element to growth factors and mitogens. In NIH3T3 fibroblasts, TCF binding is required for transcriptional activation by the SRE in response to activation of the Ras-Raf-ERK pathway. We compared the properties of three members of the TCF family, Elk-1, SAP-1 and SAP-2 (ERP/NET). Although all the proteins contain sequences required for ternary complex formation with SRF, only Elk-1 and SAP-1 appear to interact with the c-fos SRE efficiently in vivo. Each TCF contains a C-terminal activation domain capable of transcriptional activation in response to activation of the Ras-Raf-ERK pathway, and this is dependent on the integrity of S/T-P motifs conserved between all the TCF family members. In contrast, activation of the SRE by whole serum and the mitogenic phospholipid LPA requires SRF binding alone. Constitutively activated members of the Rho subfamily of Ras-like GTPases are also capable of inducing activation of the SRE in the absence of TCF; unlike activated Ras itself, these proteins do not activate the TCFs in NIH3T3 cells. At the SRE, SRF- and TCF-linked signalling pathways act synergistically to potentiate transcription.

Major immunogenic proteins of phocid herpes-viruses and their relationships to proteins of canine and feline herpesviruses.

PubMed

Harder, T C; Harder, M; de Swart, R L; Osterhaus, A D; Liess, B

1998-04-01

The immunogenic proteins of cells infected with the alpha- or the gamma-herpesvirus of seals, phocid herpesvirus-1 and -2 (PhHV-1, -2), were examined in radioimmunoprecipitation assays as a further step towards the development of a PhHV-1 vaccine. With sera obtained from convalescent seals of different species or murine monoclonal antibodies (Mabs), at least seven virus-induced glycoproteins were detected in lysates of PhHV-1-infected CrFK cells. A presumably disulphide-linked complex composed of glycoproteins of 59, 67 and 113/120 kDa, expressed on the surface of infected cells, was characterized as a major immunogenic infected cell protein of PhHV-1. This glycoprotein complex has previously been identified as the proteolytically cleavable glycoprotein B homologue of PhHV-1 (14). At least three distinct neutralization-relevant epitopes were operationally mapped, by using Mabs, on the glycoprotein B of PhHV-1. Among the infected cell proteins of the antigenically closely related feline and canine herpesvirus, the glycoprotein B equivalent proved to be the most highly conserved glycoprotein. Sera obtained from different seal species from Arctic, Antarctic, and European habitats did not precipitate uniform patterns of infected cell proteins from PhHV-1-infected cell lysates although similar titres of neutralizing antibodies were displayed. Thus, antigenic differences among the alphaherpesvirus species prevalent in the different pinniped populations cannot be excluded. PhHV-2 displayed a different pattern of infected cell proteins and only limited cross-reactivity to PhHV-1 at the protein level was detected, which is in line with its previous classification as a distinct species, based on nucleotide sequence analysis, of the gammaherpesvirus linenge. A Mab raised against PhHV-2 and specific for a major glycoprotein of 117 kDa, cross reacted with the glycoprotein B of PhHV-1. The 117-kDa glycoprotein could represent the uncleaved PhHV-2 glycoprotein B homologue.

Expression, refolding and bio-structural analysis of a tetravalent recombinant dengue envelope domain III protein for serological diagnosis.

PubMed

Combe, Maxime; Lacoux, Xavier; Martinez, Jérôme; Méjan, Odile; Luciani, Françoise; Daniel, Soizic

2017-05-01

Dengue is a mosquito-borne disease caused by four genetically and serologically related viruses that affect several millions of people. Envelope domain III (EDIII) of the viral envelope protein contains dengue virus (DENV) type-specific and DENV complex-reactive antigenic sites. Here, we describe the expression in Escherichia coli, the refolding and bio-structural analysis of envelope domain III of the four dengue serotypes as a tetravalent dengue protein (EDIIIT2), generating an attractive diagnostic candidate. In vitro refolding of denatured EDIIIT2 was performed by successive dialysis with decreasing concentrations of chaotropic reagent and in the presence of oxidized glutathione. The efficiency of refolding was demonstrated by protein mobility shifting and fluorescent visualization of labeled cysteine in non-reducing SDS-PAGE. The identity and the fully oxidized state of the protein were verified by mass spectrometry. Analysis of the structure by fluorescence, differential scanning calorimetry and circular dichroism showed a well-formed structural conformation mainly composed of β-strands. A label-free immunoassay based on biolayer interferometry technology was subsequently used to evaluate antigenic properties of folded EDIIIT2 protein using a panel of dengue IgM positive and negative human sera. Our data collectively support the use of an oxidatively refolded EDIIIT2 recombinant chimeric protein as a promising antigen in the serological diagnosis of dengue virus infections. Copyright © 2017 Elsevier Inc. All rights reserved.

Electrochemically controlled drug-mimicking protein release from iron-alginate thin-films associated with an electrode.

PubMed

Jin, Zhiyuan; Güven, Güray; Bocharova, Vera; Halámek, Jan; Tokarev, Ihor; Minko, Sergiy; Melman, Artem; Mandler, Daniel; Katz, Evgeny

2012-01-01

Novel biocompatible hybrid-material composed of iron-ion-cross-linked alginate with embedded protein molecules has been designed for the signal-triggered drug release. Electrochemically controlled oxidation of Fe(2+) ions in the presence of soluble natural alginate polymer and drug-mimicking protein (bovine serum albumin, BSA) results in the formation of an alginate-based thin-film cross-linked by Fe(3+) ions at the electrode interface with the entrapped protein. The electrochemically generated composite thin-film was characterized by electrochemistry and atomic force microscopy (AFM). Preliminary experiments demonstrated that the electrochemically controlled deposition of the protein-containing thin-film can be performed at microscale using scanning electrochemical microscopy (SECM) as the deposition tool producing polymer-patterned spots potentially containing various entrapped drugs. Application of reductive potentials on the modified electrode produced Fe(2+) cations which do not keep complexation with alginate, thus resulting in the electrochemically triggered thin-film dissolution and the protein release. Different experimental parameters, such as the film-deposition time, concentrations of compounds and applied potentials, were varied in order to demonstrate that the electrodepositon and electrodissolution of the alginate composite film can be tuned to the optimum performance. A statistical modeling technique was applied to find optimal conditions for the formation of the composite thin-film for the maximal encapsulation and release of the drug-mimicking protein at the lowest possible potential. © 2011 American Chemical Society

beta. -Amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues

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

Selkoe, D.J.; Podlisny, M.B.; Joachim, C.L.

1988-10-01

Progressive cerebral deposition of extracellular filaments composed of the {beta}-amyloid protein ({beta}AP) is a constant feature of Alzheimer disease (AD). Since the gene on chromosome 21 encoding the {beta}AP precursor ({beta}APP) is not known to be altered in AD, transcriptional or posttranslational changes may underlie accelerated {beta}AP deposition. Using two antibodies to the predicted carboxyl terminus of {beta}APP, the authors have identified the native {beta}APP in brain and nonneural human tissues as a 110- to 135-kDa protein complex that is insoluble in buffer and found in various membrane-rich subcellular fractions. These proteins are relatively uniformly distributed in adult brain, abundantmore » in fetal brain, and detected in nonneural tissues that contain {beta}APP mRNA. Similarly sized proteins occur in rat, cow, and monkey brain and in cultured human HL-60 and HeLa cells; the precise patterns in the 110- to 135-kDa range are heterogeneous among various tissues and cell lines. They conclude that the highly conserved {beta}APP molecule occurs in mammalian tissues as a heterogeneous group of membrane-associated proteins of {approx} 120 kDa. Detection of the nonamyloidogenic carboxyl terminus within plaques suggests that proteolytic processing of the {beta}APP into insoluble filaments occurs locally in cortical regions that develop {beta}-amyloid deposits with age.« less

GABA(C) receptors: a molecular view.

PubMed

Enz, R

2001-08-01

In the central nervous system inhibitory neurotransmission is primarily achieved through activation of receptors for gamma-aminobutyric acid (GABA). Three types of GABA receptors have been identified on the basis of their pharmacological and electrophysiological properties. The predominant type, termed GABA(A), and a recently identified GABA(C) type, form ligand-gated chloride channels, whereas GABA(B) receptors activate separate cation channels via G proteins. Based on their homology to nicotinic acetylcholine receptors, GABA(C) receptors are believed to be oligomeric protein complexes composed of five subunits in a pentameric arrangement. To date up to five different GABA(C) receptors subunits have been identified in various species. Recent studies have shed new light on the biological characteristics of GABA(C) receptors, including the chromosomal localization of its subunit genes and resulting links to deseases, the cloning of new splice variants, the identification of GABA(C) receptor-associated proteins, the identification of domains involved in subunit assembly, and finally structure/function studies examining functional consequences of introduced mutations. This review summarizes recent data in view of the molecular structure of GABA(C) receptors and presents new insights into the biological function of this protein in the retina.

A Role for Timely Nuclear Translocation of Clock Repressor Proteins in Setting Circadian Clock Speed

PubMed Central

Lee, Euna

2014-01-01

By means of a circadian clock system, all the living organisms on earth including human beings can anticipate the environmental rhythmic changes such as light/dark and warm/cold periods in a daily as well as in a yearly manner. Anticipating such environmental changes provide organisms with survival benefits via manifesting behavior and physiology at an advantageous time of the day and year. Cell-autonomous circadian oscillators, governed by transcriptional feedback loop composed of positive and negative elements, are organized into a hierarchical system throughout the organisms and generate an oscillatory expression of a clock gene by itself as well as clock controlled genes (ccgs) with a 24 hr periodicity. In the feedback loop, hetero-dimeric transcription factor complex induces the expression of negative regulatory proteins, which in turn represses the activity of transcription factors to inhibit their own transcription. Thus, for robust oscillatory rhythms of the expression of clock genes as well as ccgs, the precise control of subcellular localization and/or timely translocation of core clock protein are crucial. Here, we discuss how sub-cellular localization and nuclear translocation are controlled in a time-specific manner focusing on the negative regulatory clock proteins. PMID:25258565

Bioorthogonal Modification of the Major Sheath Protein of Bacteriophage M13: Extending the Versatility of Bionanomaterial Scaffolds.

PubMed

Urquhart, Taylor; Daub, Elisabeth; Honek, John Frank

2016-10-19

With a mass of ∼1.6 × 10 7 Daltons and composed of approximately 2700 proteins, bacteriophage M13 has been employed as a molecular scaffold in bionanomaterials fabrication. In order to extend the versatility of M13 in this area, residue-specific unnatural amino acid incorporation was employed to successfully display azide functionalities on specific solvent-exposed positions of the pVIII major sheath protein of this bacteriophage. Employing a combination of engineered mutants of the gene coding for the pVIII protein, the methionine (Met) analog, l-azidohomoalanine (Aha), and a suitable Escherichia coli Met auxotroph for phage production, conditions were developed to produce M13 bacteriophage labeled with over 350 active azides (estimated by fluorescent dye labeling utilizing a strain-promoted azide-alkyne cycloaddition) and capable of azide-selective attachment to 5 nm gold nanoparticles as visualized by transmission electron microscopy. The capability of this system to undergo dual labeling utilizing both chemical acylation and bioorthogonal cycloaddition reactions was also verified. The above stratagem should prove particularly advantageous in the preparation of assemblies of larger and more complex molecular architectures based on the M13 building block.

Sunlight mediated synthesis of silver nanoparticles using redox phytoprotein and their application in catalysis and colorimetric mercury sensing.

PubMed

Ahmed, Khan Behlol Ayaz; Senthilnathan, Rajendran; Megarajan, Sengan; Anbazhagan, Veerappan

2015-10-01

Owing to the benign nature, plant extracts mediated green synthesis of metal nanoparticles (NPs) is rapidly expanding. In this study, we demonstrated the successful green synthesis of silver nanoparticles (AgNPs) by utilizing natural sunlight and redox protein complex composed of ferredoxin-NADP(+) reductase (FNR) and ferredoxin (FD). The capping and stabilization of the AgNPs by the redox protein was confirmed by Fourier transform infrared spectroscopy. Light and redox protein is the prerequisite factor for the formation of AgNPs. The obtained result shows that the photo generated free radicals by the redox protein is responsible for the reduction of Ag(+) to Ag(0). Transmission electron microscopy revealed the formation of spherical AgNPs with size ranging from 10 to 15 nm. As-prepared AgNPs exhibit excellent catalytic activity toward the degradation of hazardous organic dyes, such as methylene blue, methyl orange and methyl red. These bio-inspired AgNPs is highly sensitive and selective in sensing hazardous mercury ions in the water at micromolar concentration. In addition, FNR/FD extract stabilized AgNPs showed good antimicrobial activity against gram positive and gram negative bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Rasmussen, Søren G.F.; DeVree, Brian T; Zou, Yaozhong

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β 2 adrenergic receptor (β 2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β 2AR and nucleotide-free Gs heterotrimer. The principal interactions between the βmore » 2AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β 2AR include a 14Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.« less

Nanobiotechnology with S-layer proteins as building blocks.

PubMed

Sleytr, Uwe B; Schuster, Bernhard; Egelseer, Eva M; Pum, Dietmar; Horejs, Christine M; Tscheliessnig, Rupert; Ilk, Nicola

2011-01-01

One of the key challenges in nanobiotechnology is the utilization of self- assembly systems, wherein molecules spontaneously associate into reproducible aggregates and supramolecular structures. In this contribution, we describe the basic principles of crystalline bacterial surface layers (S-layers) and their use as patterning elements. The broad application potential of S-layers in nanobiotechnology is based on the specific intrinsic features of the monomolecular arrays composed of identical protein or glycoprotein subunits. Most important, physicochemical properties and functional groups on the protein lattice are arranged in well-defined positions and orientations. Many applications of S-layers depend on the capability of isolated subunits to recrystallize into monomolecular arrays in suspension or on suitable surfaces (e.g., polymers, metals, silicon wafers) or interfaces (e.g., lipid films, liposomes, emulsomes). S-layers also represent a unique structural basis and patterning element for generating more complex supramolecular structures involving all major classes of biological molecules (e.g., proteins, lipids, glycans, nucleic acids, or combinations of these). Thus, S-layers fulfill key requirements as building blocks for the production of new supramolecular materials and nanoscale devices as required in molecular nanotechnology, nanobiotechnology, biomimetics, and synthetic biology. Copyright © 2011 Elsevier Inc. All rights reserved.

MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity

PubMed Central

Patron, Maria; Checchetto, Vanessa; Raffaello, Anna; Teardo, Enrico; Vecellio Reane, Denis; Mantoan, Maura; Granatiero, Veronica; Szabò, Ildikò; De Stefani, Diego; Rizzuto, Rosario

2014-01-01

Summary Mitochondrial calcium accumulation was recently shown to depend on a complex composed of an inner-membrane channel (MCU and MCUb) and regulatory subunits (MICU1, MCUR1, and EMRE). A fundamental property of MCU is low activity at resting cytosolic Ca2+ concentrations, preventing deleterious Ca2+ cycling and organelle overload. Here we demonstrate that these properties are ensured by a regulatory heterodimer composed of two proteins with opposite effects, MICU1 and MICU2, which, both in purified lipid bilayers and in intact cells, stimulate and inhibit MCU activity, respectively. Both MICU1 and MICU2 are regulated by calcium through their EF-hand domains, thus accounting for the sigmoidal response of MCU to [Ca2+] in situ and allowing tight physiological control. At low [Ca2+], the dominant effect of MICU2 largely shuts down MCU activity; at higher [Ca2+], the stimulatory effect of MICU1 allows the prompt response of mitochondria to Ca2+ signals generated in the cytoplasm. PMID:24560927

Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

PubMed Central

Martorana, Alessandra M.; Benedet, Mattia; Maccagni, Elisa A.; Sperandeo, Paola; Villa, Riccardo; Dehò, Gianni

2016-01-01

ABSTRACT The assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made in Escherichia coli of seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level of lptB is a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carries Pa-LptC, the highly divergent LptC orthologue from Pseudomonas aeruginosa. We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carrying Pa-LptC which is otherwise defective in interactions with the E. coli LptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery. IMPORTANCE The asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. In Escherichia coli the LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a transenvelope complex. Here, we show that increased expression of the membrane-associated ABC protein LptB can suppress defects of LptC, which participates in the formation of the periplasmic bridge. This reveals functional interactions between these two components and supports a role of LptB in the assembly of the Lpt machine. PMID:27246575

"And Now That I Know Them": Composing Mutuality in a Service Learning Course.

ERIC Educational Resources Information Center

Welch, Nancy

2002-01-01

Turns to contemporary feminist object-relations theory to understand the efforts of students in a service learning course, to push beyond the usual subject-object, active-passive dualisms that pervade community-based literacy projects, and to compose instead complex representations in which all participants are composed as active, as knowing, and…

Computational redesign of a protein-protein interface for high affinity and binding specificity using modular architecture and naturally occurring template fragments.

PubMed

Potapov, V; Reichmann, D; Abramovich, R; Filchtinski, D; Zohar, N; Ben Halevy, D; Edelman, M; Sobolev, V; Schreiber, G

2008-12-05

A new method is presented for the redesign of protein-protein interfaces, resulting in specificity of the designed pair while maintaining high affinity. The design is based on modular interface architecture and was carried out on the interaction between TEM1 beta-lactamase and its inhibitor protein, beta-lactamase inhibitor protein. The interface between these two proteins is composed of several mostly independent modules. We previously showed that it is possible to delete a complete module without affecting the overall structure of the interface. Here, we replace a complete module with structure fragments taken from nonrelated proteins. Nature-optimized fragments were chosen from 10(7) starting templates found in the Protein Data Bank. A procedure was then developed to identify sets of interacting template residues with a backbone arrangement mimicking the original module. This generated a final list of 361 putative replacement modules that were ranked using a novel scoring function based on grouped atom-atom contact surface areas. The top-ranked designed complex exhibited an affinity of at least the wild-type level and a mode of binding that was remarkably specific despite the absence of negative design in the procedure. In retrospect, the combined application of three factors led to the success of the design approach: utilizing the modular construction of the interface, capitalizing on native rather than artificial templates, and ranking with an accurate atom-atom contact surface scoring function.

Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis

PubMed Central

McKenzie, Callum; Bassi, Zuni I.; Debski, Janusz; Gottardo, Marco; Callaini, Giuliano; Dadlez, Michal; D'Avino, Pier Paolo

2016-01-01

Cytokinesis culminates in the final separation, or abscission, of the two daughter cells at the end of cell division. Abscission relies on an organelle, the midbody, which forms at the intercellular bridge and is composed of various proteins arranged in a precise stereotypic pattern. The molecular mechanisms controlling midbody organization and function, however, are obscure. Here we show that proper midbody architecture requires cross-regulation between two cell division kinases, Citron kinase (CIT-K) and Aurora B, the kinase component of the chromosomal passenger complex (CPC). CIT-K interacts directly with three CPC components and is required for proper midbody architecture and the orderly arrangement of midbody proteins, including the CPC. In addition, we show that CIT-K promotes Aurora B activity through phosphorylation of the INCENP CPC subunit at the TSS motif. In turn, Aurora B controls CIT-K localization and association with its central spindle partners through phosphorylation of CIT-K's coiled coil domain. Our results identify, for the first time, a cross-regulatory mechanism between two kinases during cytokinesis, which is crucial for establishing the stereotyped organization of midbody proteins. PMID:27009191

Usher syndrome type 1–associated cadherins shape the photoreceptor outer segment

PubMed Central

Parain, Karine; Aghaie, Asadollah; Picaud, Serge

2017-01-01

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15–containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal. PMID:28495838

Usher syndrome type 1-associated cadherins shape the photoreceptor outer segment.

PubMed

Schietroma, Cataldo; Parain, Karine; Estivalet, Amrit; Aghaie, Asadollah; Boutet de Monvel, Jacques; Picaud, Serge; Sahel, José-Alain; Perron, Muriel; El-Amraoui, Aziz; Petit, Christine

2017-06-05

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis , these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23 , encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15-containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal. © 2017 Schietroma et al.

Insight into the Sialome of the Bed Bug, Cimex lectularius.

PubMed

Francischetti, Ivo M B; Calvo, Eric; Andersen, John F; Pham, Van M; Favreau, Amanda J; Barbian, Kent D; Romero, Alvaro; Valenzuela, Jesus G; Ribeiro, José M C

2010-08-06

The evolution of insects to a blood diet leads to the development of a saliva that antagonizes their hosts' hemostasis and inflammation. Hemostasis and inflammation are redundant processes, and thus a complex salivary potion composed of dozens or near 100 different polypeptides is commonly found by transcriptome or proteome analysis of these organisms. Several insect orders or families evolved independently to hematophagy, creating unique salivary potions in the form of novel pharmacological use of endogenous substances and in the form of unique proteins not matching other known proteins, these probably arriving by fast evolution of salivary proteins as they evade their hosts' immune response. In this work we present a preliminary description of the sialome (from the Greek Sialo = saliva) of the common bed bug Cimex lectularius, the first such work from a member of the Cimicidae family. This manuscript is a guide for the supplemental database files http://exon.niaid.nih.gov/transcriptome/C_lectularius/S1/Cimex-S1.zip and http://exon.niaid.nih.gov/transcriptome/C_lectularius/S2/Cimex-S2.xls.

Demonstration of a Specific Escherichia coli SecY–Signal Peptide Interaction†

PubMed Central

Wang, Ligong; Miller, Alexander; Rusch, Sharyn L.; Kendall, Debra A.

2011-01-01

Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide–SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA. PMID:15476412

Structure and membrane remodeling activity of ESCRT-III helical polymers

DOE PAGES

McCullough, John; Clippinger, Amy K.; Talledge, Nathaniel; ...

2015-12-18

The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecturemore » and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. In conclusion, our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.« less

The Adaptor Protein SAP Directly Associates with CD3ζ Chain and Regulates T Cell Receptor Signaling

PubMed Central

Proust, Richard; Bertoglio, Jacques; Gesbert, Franck

2012-01-01

Mutations altering the gene encoding the SLAM associated protein (SAP) are responsible for the X-linked lymphoproliferative disease or XLP1. Its absence is correlated with a defective NKT cells development, a decrease in B cell functions and a reduced T cells and NK cells cytotoxic activities, thus leading to an immunodeficiency syndrome. SAP is a small 128 amino-acid long protein that is almost exclusively composed of an SH2 domain. It has been shown to interact with the CD150/SLAM family of receptors, and in a non-canonical manner with SH3 containing proteins such as Fyn, βPIX, PKCθ and Nck1. It would thus play the role of a minimal adaptor protein. It has been shown that SAP plays an important function in the activation of T cells through its interaction with the SLAM family of receptors. Therefore SAP defective T cells display a reduced activation of signaling events downstream of the TCR-CD3 complex triggering. In the present work, we evidence that SAP is a direct interactor of the CD3ζ chain. This direct interaction occurs through the first ITAM of CD3ζ, proximal to the membrane. Additionally, we show that, in the context of the TCR-CD3 signaling, an Sh-RNA mediated silencing of SAP is responsible for a decrease of several canonical T cell signaling pathways including Erk, Akt and PLCγ1 and to a reduced induction of IL-2 and IL-4 mRNA. Altogether, we show that SAP plays a central function in the T cell activation processes through a direct association with the CD3 complex. PMID:22912825

Evolution of Antifreeze Protein Genes in the Diatom Genus Fragilariopsis: Evidence for Horizontal Gene Transfer, Gene Duplication and Episodic Diversifying Selection

PubMed Central

Sorhannus, Ulf

2011-01-01

Hypotheses about horizontal transfer of antifreeze protein genes to ice-living diatoms were addressed using two different statistical methods available in the program Prunier. The role of diversifying selection in driving the differentiation of a set of antifreeze protein genes in the diatom genus Fragilariopsis was also investigated. Four horizontal gene transfer events were identified. Two of these took place between two major eukaryote lineages, that is from the diatom Chaetoceros neogracile to the copepod Stephos longipes and from a basidiomycete clade to a monophyletic group, consisting of the diatom species Fragilariopsis curta and Fragilariopsis cylindrus. The remaining two events included transfers from an ascomycete lineage to the proteobacterium Stigmatella aurantiaca and from the proteobacterium Polaribacter irgensii to a group composed of 4 proteobacterium species. After the Fragilariopsis lineage acquired the antifreeze protein gene from the basidiomycetes, it duplicated and went through episodic evolution, characterized by strong positive selection acting on short segments of the branches in the tree. This selection pattern suggests that the paralogs differentiated functionally over relatively short time periods. Taken together, the results obtained here indicate that the group of antifreeze protein genes considered here have a complex evolutionary history. PMID:22253534

Structure and function of gap junction proteins: role of gap junction proteins in embryonic heart development.

PubMed

Ahir, Bhavesh K; Pratten, Margaret K

2014-01-01

Intercellular (cell-to-cell) communication is a crucial and complex mechanism during embryonic heart development. In the cardiovascular system, the beating of the heart is a dynamic and key regulatory process, which is functionally regulated by the coordinated spread of electrical activity through heart muscle cells. Heart tissues are composed of individual cells, each bearing specialized cell surface membrane structures called gap junctions that permit the intercellular exchange of ions and low molecular weight molecules. Gap junction channels are essential in normal heart function and they assist in the mediated spread of electrical impulses that stimulate synchronized contraction (via an electrical syncytium) of cardiac tissues. This present review describes the current knowledge of gap junction biology. In the first part, we summarise some relevant biochemical and physiological properties of gap junction proteins, including their structure and function. In the second part, we review the current evidence demonstrating the role of gap junction proteins in embryonic development with particular reference to those involved in embryonic heart development. Genetics and transgenic animal studies of gap junction protein function in embryonic heart development are considered and the alteration/disruption of gap junction intercellular communication which may lead to abnormal heart development is also discussed.

Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins

PubMed Central

De Jaco, Antonella; Dubi, Noga; Camp, Shelley; Taylor, Palmer

2017-01-01

The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination. PMID:23035660

Fabrication of diverse pH-sensitive functional mesoporous silica for selective removal or depletion of highly abundant proteins from biological samples.

PubMed

Wang, Jiaojiao; Lan, Jingfeng; Li, Huihui; Liu, Xiaoyan; Zhang, Haixia

2017-01-01

In proteomic studies, poor detection of low abundant proteins is a major problem due to the presence of highly abundant proteins. Therefore, the specific removal or depletion of highly abundant proteins prior to analysis is necessary. In response to this problem, a series of pH-sensitive functional mesoporous silica materials composed of 2-(diethylamino)ethyl methacrylate and methacrylic acid units were designed and synthesized via atom transfer radical polymerization. These functional mesoporous silica materials were characterized and their ability for adsorption and separation of proteins was evaluated. Possessing a pH-sensitive feature, the synthesized functional materials showed selective adsorption of some proteins in aqueous or buffer solutions at certain pH values. The specific removal of a particular protein from a mixed protein solution was subsequently studied. The analytical results confirmed that all the target proteins (bovine serum albumin, ovalbumin, and lysozyme) can be removed by the proposed materials from a five-protein mixture in a single operation. Finally, the practical application of this approach was also evaluated by the selective removal of certain proteins from real biological samples. The results revealed that the maximum removal efficiencies of ovalbumin and lysozyme from egg white sample were obtained as 99% and 92%, respectively, while the maximum removal efficiency of human serum albumin from human serum sample was about 80% by the proposed method. It suggested that this treatment process reduced the complexity of real biological samples and facilitated the identification of hidden proteins in chromatograms. Copyright © 2016 Elsevier B.V. All rights reserved.

Construction of protocellular structures under simulated primitive earth conditions

NASA Astrophysics Data System (ADS)

Yanagawa, Hiroshi; Ogawa, Yoko; Kojima, Kiyotsugu; Ito, Masahiko

1988-09-01

We have developed experimental approaches for the construction of protocellular structures under simulated primitive earth conditions and studied their formation and characteristics. Three types of envelopes; protein envelopes, lipid envelopes, and lipid-protein envelopes are considered as candidates for protocellular structures. Simple protein envelopes and lipid envelopes are presumed to have originated at an early stage of chemical evolution, interaction mutually and then evolved into more complex envelopes composed of both lipids and proteins. Three kinds of protein envelopes were constructedin situ from amino acids under simulated primitive earth conditions such as a fresh water tide pool, a warm sea, and a submarine hydrothermal vent. One protein envelope was formed from a mixture of amino acid amides at 80 °C using multiple hydration-dehydration cycles. Marigranules, protein envelope structures, were produced from mixtures of glycine and acidic, basic and aromatic amino acids at 105 °C in a modified sea medium enriched with essential transition elements. Thermostable microspheres were also formed from a mixture of glycine, alanine, valine, and aspartic acid at 250 °C and above. The microspheres did not form at lower temperatures and consist of silicates and peptide-like polymers containing imide bonds and amino acid residues enriched in valine. Amphiphilic proteins with molecular weights of 2000 were necessary for the formation of the protein envelopes. Stable lipid envelopes were formed from different dialkyl phospholipids and fatty acids. Large, stable, lipid-protein envelopes were formed from egg lecithin and the solubilized marigranules. Polycations such as polylysine and polyhistidine, or basic proteins such as lysozyme and cytochromec also stabilized lipid-protein envelopes.

The mate recognition protein gene mediates reproductive isolation and speciation in the Brachionus plicatilis cryptic species complex

PubMed Central

2012-01-01

Background Chemically mediated prezygotic barriers to reproduction likely play an important role in speciation. In facultatively sexual monogonont rotifers from the Brachionus plicatilis cryptic species complex, mate recognition of females by males is mediated by the Mate Recognition Protein (MRP), a globular glycoprotein on the surface of females, encoded by the mmr-b gene family. In this study, we sequenced mmr-b copies from 27 isolates representing 11 phylotypes of the B. plicatilis species complex, examined the mode of evolution and selection of mmr-b, and determined the relationship between mmr-b genetic distance and mate recognition among isolates. Results Isolates of the B. plicatilis species complex have 1–4 copies of mmr-b, each composed of 2–9 nearly identical tandem repeats. The repeats within a gene copy are generally more similar than are gene copies among phylotypes, suggesting concerted evolution. Compared to housekeeping genes from the same isolates, mmr-b has accumulated only half as many synonymous differences but twice as many non-synonymous differences. Most of the amino acid differences between repeats appear to occur on the outer face of the protein, and these often result in changes in predicted patterns of phosphorylation. However, we found no evidence of positive selection driving these differences. Isolates with the most divergent copies were unable to mate with other isolates and rarely self-crossed. Overall the degree of mate recognition was significantly correlated with the genetic distance of mmr-b. Conclusions Discrimination of compatible mates in the B. plicatilis species complex is determined by proteins encoded by closely related copies of a single gene, mmr-b. While concerted evolution of the tandem repeats in mmr-b may function to maintain identity, it can also lead to the rapid spread of a mutation through all copies in the genome and thus to reproductive isolation. The mmr-b gene is evolving rapidly, and novel alleles may be maintained and increase in frequency via asexual reproduction. Our analyses indicate that mate recognition, controlled by MMR-B, may drive reproductive isolation and allow saltational sympatric speciation within the B. plicatilis cryptic species complex, and that this process may be largely neutral. PMID:22852831

Benchmarking quantitative label-free LC-MS data processing workflows using a complex spiked proteomic standard dataset.

PubMed

Ramus, Claire; Hovasse, Agnès; Marcellin, Marlène; Hesse, Anne-Marie; Mouton-Barbosa, Emmanuelle; Bouyssié, David; Vaca, Sebastian; Carapito, Christine; Chaoui, Karima; Bruley, Christophe; Garin, Jérôme; Cianférani, Sarah; Ferro, Myriam; Van Dorssaeler, Alain; Burlet-Schiltz, Odile; Schaeffer, Christine; Couté, Yohann; Gonzalez de Peredo, Anne

2016-01-30

Proteomic workflows based on nanoLC-MS/MS data-dependent-acquisition analysis have progressed tremendously in recent years. High-resolution and fast sequencing instruments have enabled the use of label-free quantitative methods, based either on spectral counting or on MS signal analysis, which appear as an attractive way to analyze differential protein expression in complex biological samples. However, the computational processing of the data for label-free quantification still remains a challenge. Here, we used a proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate to benchmark several label-free quantitative workflows, involving different software packages developed in recent years. This experimental design allowed to finely assess their performances in terms of sensitivity and false discovery rate, by measuring the number of true and false-positive (respectively UPS1 or yeast background proteins found as differential). The spiked standard dataset has been deposited to the ProteomeXchange repository with the identifier PXD001819 and can be used to benchmark other label-free workflows, adjust software parameter settings, improve algorithms for extraction of the quantitative metrics from raw MS data, or evaluate downstream statistical methods. Bioinformatic pipelines for label-free quantitative analysis must be objectively evaluated in their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. This can be done through the use of complex spiked samples, for which the "ground truth" of variant proteins is known, allowing a statistical evaluation of the performances of the data processing workflow. We provide here such a controlled standard dataset and used it to evaluate the performances of several label-free bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold) in different workflows, for detection of variant proteins with different absolute expression levels and fold change values. The dataset presented here can be useful for tuning software tool parameters, and also testing new algorithms for label-free quantitative analysis, or for evaluation of downstream statistical methods. Copyright © 2015 Elsevier B.V. All rights reserved.

The interaction between the yeast telomerase RNA and the Est1 protein requires three structural elements.

PubMed

Lubin, Johnathan W; Tucey, Timothy M; Lundblad, Victoria

2012-09-01

In the budding yeast Saccharomyces cerevisiae, the telomerase enzyme is composed of a 1.3-kb TLC1 RNA that forms a complex with Est2 (the catalytic subunit) and two regulatory proteins, Est1 and Est3. Previous work has identified a conserved 5-nt bulge, present in a long helical arm of TLC1, which mediates binding of Est1 to TLC1. However, increased expression of Est1 can bypass the consequences of removal of this RNA bulge, indicating that there are additional binding site(s) for Est1 on TLC1. We report here that a conserved single-stranded internal loop immediately adjacent to the bulge is also required for the Est1-RNA interaction; furthermore, a TLC1 variant that lacks this internal loop but retains the bulge cannot be suppressed by Est1 overexpression, arguing that the internal loop may be a more critical element for Est1 binding. An additional structural feature consisting of a single-stranded region at the base of the helix containing the bulge and internal loop also contributes to recognition of TLC1 by Est1, potentially by providing flexibility to this helical arm. Association of Est1 with each of these TLC1 motifs was assessed using a highly sensitive biochemical assay that simultaneously monitors the relative levels of the Est1 and Est2 proteins in the telomerase complex. The identification of three elements of TLC1 that are required for Est1 association provides a detailed view of this particular protein-RNA interaction.

cpSRP43 Is a Novel Chaperone Specific for Light-harvesting Chlorophyll a,b-binding Proteins*

PubMed Central

Falk, Sebastian; Sinning, Irmgard

2010-01-01

The biosynthesis of most membrane proteins is directly coupled to membrane insertion, and therefore, molecular chaperones are not required. The light-harvesting chlorophyll a,b-binding proteins (LHCPs) present a prominent exception as they are synthesized in the cytoplasm, and after import into the chloroplast, they are targeted and inserted into the thylakoid membrane. Upon arrival in the stroma, LHCPs form a soluble transit complex with the chloroplast signal recognition particle (cpSRP) consisting of an SRP54 homolog and the unique cpSRP43 composed of three chromodomains and four ankyrin repeats. Here we describe that cpSRP43 alone prevents aggregation of LHCP by formation of a complex with nanomolar affinity, whereas cpSRP54 is not required for this chaperone activity. Other stromal chaperones like trigger factor cannot replace cpSRP43, which implies that LHCPs require a specific chaperone. Although cpSRP43 does not have an ATPase activity, it can dissolve aggregates of LHCPs similar to chaperones of the Hsp104/ClpB family. We show that the LHCP-cpSRP43 interaction is predominantly hydrophobic but strictly depends on an intact DPLG motif between the second and third transmembrane region. The cpSRP43 ankyrin repeats that provide the binding site for the DPLG motif are sufficient for the chaperone function, whereas the chromodomains are dispensable. Taken together, we define cpSRP43 as a highly specific chaperone for LHCPs in addition to its established function as a targeting factor for this family of membrane proteins. PMID:20498370

Multi-branched ionic liquid-chitosan as a smart and biocompatible nano-vehicle for combination chemotherapy with stealth and targeted properties.

PubMed

Rahimi, Mahdi; Shafiei-Irannejad, Vahid; D Safa, Kazem; Salehi, Roya

2018-09-15

A possible approach for clinical cancer treatment is combination chemotherapy. To address this issue, many anticancer agents have been used simultaneously to achieve synergistic effects with the different mechanism of actions, however, their toxic side effects are still a big challenge. In this study, a smart, biocompatible, magnetic nanocarrier composed of multi-branched ionic liquid-chitosan grafted mPEG was designed and used for targeted multidrug delivery of DOX and MTX as model anticancer agents to MCF7 breast cancer cells. The results of hemolysis assay on human red blood cells and cytotoxicity studies indicated that blank nanocarrier has no significant hemolytic and cytotoxic effects in MCF7 cells as observed in the results of MTT assay, however, drugs-loaded nanocarrier could decrease the viability of MCF7 cells in a dose-dependent manner. To further simulate the interaction of nanocarrier with plasma proteins, the SDS-PAGE assay was performed after the nanocarrier was incubated with human plasma and the results indicated that a series of proteins were attached to the surface of nanocarrier leading protein-particle corona complex. This complex gives a stealth property as well as increasing cellular uptake process due to the presence of proteins acting as ligands for receptors in the surface of cancer cells that are suitable for drug delivery systems. The efficiency of dual-drug delivery was also confirmed by cellular uptake and DAPI staining. All these results persuade us, this nanocarrier is suitable for use in further animal studies in future investigations. Copyright © 2018 Elsevier Ltd. All rights reserved.

Initial characterization of the FlgE hook high molecular weight complex of Borrelia burgdorferi.

PubMed

Miller, Kelly A; Motaleb, Md A; Liu, Jun; Hu, Bo; Caimano, Melissa J; Miller, Michael R; Charon, Nyles W

2014-01-01

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.

Initial Characterization of the FlgE Hook High Molecular Weight Complex of Borrelia burgdorferi

PubMed Central

Miller, Kelly A.; Motaleb, Md. A.; Liu, Jun; Hu, Bo; Caimano, Melissa J.; Miller, Michael R.; Charon, Nyles W.

2014-01-01

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility. PMID:24859001

Hybrid- and complex-type N-glycans are not essential for Newcastle disease virus infection and fusion of host cells.

PubMed

Sun, Qing; Zhao, Lixiang; Song, Qingqing; Wang, Zheng; Qiu, Xusheng; Zhang, Wenjun; Zhao, Mingjun; Zhao, Guo; Liu, Wenbo; Liu, Haiyan; Li, Yunsen; Liu, Xiufan

2012-03-01

N-linked glycans are composed of three major types: high-mannose (Man), hybrid or complex. The functional role of hybrid- and complex-type N-glycans in Newcastle disease virus (NDV) infection and fusion was examined in N-acetylglucosaminyltransferase I (GnT I)-deficient Lec1 cells, a mutant Chinese hamster ovary (CHO) cell incapable of synthesizing hybrid- and complex-type N-glycans. We used recombinant NDV expressing green fluorescence protein or red fluorescence protein to monitor NDV infection, syncytium formation and viral yield. Flow cytometry showed that CHO-K1 and Lec1 cells had essentially the same degree of NDV infection. In contrast, Lec2 cells were found to be resistant to NDV infection. Compared with CHO-K1 cells, Lec1 cells were shown to more sensitive to fusion induced by NDV. Viral attachment was found to be comparable in both lines. We found that there were no significant differences in the yield of progeny virus produced by both CHO-K1 and Lec1 cells. Quantitative analysis revealed that NDV infection and fusion in Lec1 cells were also inhibited by treatment with sialidase. Pretreatment of Lec1 cells with Galanthus nivalis agglutinin specific for terminal α1-3-linked Man prior to inoculation with NDV rendered Lec1 cells less sensitive to cell-to-cell fusion compared with mock-treated Lec1 cells. Treatment of CHO-K1 and Lec1 cells with tunicamycin, an inhibitor of N-glycosylation, significantly blocked fusion and infection. In conclusion, our results suggest that hybrid- and complex-type N-glycans are not required for NDV infection and fusion. We propose that high-Man-type N-glycans could play an important role in the cell-to-cell fusion induced by NDV.

The Crystal Structure of the Ubiquitin-like Domain of Ribosome Assembly Factor Ytm1 and Characterization of Its Interaction with the AAA-ATPase Midasin*

PubMed Central

Romes, Erin M.; Sobhany, Mack; Stanley, Robin E.

2016-01-01

The synthesis of eukaryotic ribosomes is a complex, energetically demanding process requiring the aid of numerous non-ribosomal factors, such as the PeBoW complex. The mammalian PeBoW complex, composed of Pes1, Bop1, and WDR12, is essential for the processing of the 32S preribosomal RNA. Previous work in Saccharomyces cerevisiae has shown that release of the homologous proteins in this complex (Nop7, Erb1, and Ytm1, respectively) from preribosomal particles requires Rea1 (midasin or MDN1 in humans), a large dynein-like protein. Midasin contains a C-terminal metal ion-dependent adhesion site (MIDAS) domain that interacts with the N-terminal ubiquitin-like (UBL) domain of Ytm1/WDR12 as well as the UBL domain of Rsa4/Nle1 in a later step in the ribosome maturation pathway. Here we present the crystal structure of the UBL domain of the WDR12 homologue from S. cerevisiae at 1.7 Å resolution and demonstrate that human midasin binds to WDR12 as well as Nle1 through their respective UBL domains. Midasin contains a well conserved extension region upstream of the MIDAS domain required for binding WDR12 and Nle1, and the interaction is dependent upon metal ion coordination because removal of the metal or mutation of residues that coordinate the metal ion diminishes the interaction. Mammalian WDR12 displays prominent nucleolar localization that is dependent upon active ribosomal RNA transcription. Based upon these results, we propose that release of the PeBoW complex and subsequent release of Nle1 by midasin is a well conserved step in the ribosome maturation pathway in both yeast and mammalian cells. PMID:26601951

Analysis of Protein Composition and Bioactivity of Neoponera villosa Venom (Hymenoptera: Formicidae).

PubMed

Pessoa, Wallace Felipe Blohem; Silva, Ludimilla Carvalho Cerqueira; de Oliveira Dias, Leila; Delabie, Jacques Hubert Charles; Costa, Helena; Romano, Carla Cristina

2016-04-21

Ants cause a series of accidents involving humans. Such accidents generate different reactions in the body, ranging from a mild irritation at the bite site to anaphylactic shock, and these reactions depend on the mechanism of action of the venom. The study of animal venom is a science known as venomics. Through venomics, the composition of the venom of several ant species has already been characterized and their biological activities described. Thus, the aim of this study was to evaluate the protein composition and biological activities (hemolytic and immunostimulatory) of the venom of Neoponera villosa (N. villosa), an ant widely distributed in South America. The protein composition was evaluated by proteomic techniques, such as two-dimensional electrophoresis. To assess the biological activity, hemolysis assay was carried out and cytokines were quantified after exposure of macrophages to the venom. The venom of N. villosa has a profile composed of 145 proteins, including structural and metabolic components (e.g., tubulin and ATPase), allergenic and immunomodulatory proteins (arginine kinase and heat shock proteins (HSPs)), protective proteins of venom (superoxide dismutase (SOD) and catalase) and tissue degradation proteins (hyaluronidase and phospholipase A2). The venom was able to induce hemolysis in human erythrocytes and also induced release of both pro-inflammatory cytokines, as the anti-inflammatory cytokine release by murine macrophages. These results allow better understanding of the composition and complexity of N. villosa venom in the human body, as well as the possible mechanisms of action after the bite.

Analysis of Protein Composition and Bioactivity of Neoponera villosa Venom (Hymenoptera: Formicidae)

PubMed Central

Pessoa, Wallace Felipe Blohem; Silva, Ludimilla Carvalho Cerqueira; de Oliveira Dias, Leila; Delabie, Jacques Hubert Charles; Costa, Helena; Romano, Carla Cristina

2016-01-01

Ants cause a series of accidents involving humans. Such accidents generate different reactions in the body, ranging from a mild irritation at the bite site to anaphylactic shock, and these reactions depend on the mechanism of action of the venom. The study of animal venom is a science known as venomics. Through venomics, the composition of the venom of several ant species has already been characterized and their biological activities described. Thus, the aim of this study was to evaluate the protein composition and biological activities (hemolytic and immunostimulatory) of the venom of Neoponera villosa (N. villosa), an ant widely distributed in South America. The protein composition was evaluated by proteomic techniques, such as two-dimensional electrophoresis. To assess the biological activity, hemolysis assay was carried out and cytokines were quantified after exposure of macrophages to the venom. The venom of N. villosa has a profile composed of 145 proteins, including structural and metabolic components (e.g., tubulin and ATPase), allergenic and immunomodulatory proteins (arginine kinase and heat shock proteins (HSPs)), protective proteins of venom (superoxide dismutase (SOD) and catalase) and tissue degradation proteins (hyaluronidase and phospholipase A2). The venom was able to induce hemolysis in human erythrocytes and also induced release of both pro-inflammatory cytokines, as the anti-inflammatory cytokine release by murine macrophages. These results allow better understanding of the composition and complexity of N. villosa venom in the human body, as well as the possible mechanisms of action after the bite. PMID:27110765

Structural and Biochemical Insights into MLL1 Core Complex Assembly

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

Avdic, Vanja; Zhang, Pamela; Lanouette, Sylvain

2012-05-02

Histone H3 Lys-4 methylation is predominantly catalyzed by a family of methyltransferases whose enzymatic activity depends on their interaction with a three-subunit complex composed of WDR5, RbBP5, and Ash2L. Here, we report that a segment of 50 residues of RbBP5 bridges the Ash2L C-terminal domain to WDR5. The crystal structure of WDR5 in ternary complex with RbBP5 and MLL1 reveals that both proteins binds peptide-binding clefts located on opposite sides of WDR5s {beta}-propeller domain. RbBP5 engages in several hydrogen bonds and van der Waals contacts within a V-shaped cleft formed by the junction of two blades on WDR5. Mutational analysesmore » of both the WDR5 V-shaped cleft and RbBP5 residues reveal that the interactions between RbBP5 and WDR5 are important for the stimulation of MLL1 methyltransferase activity. Overall, this study provides the structural basis underlying the formation of the WDR5-RbBP5 subcomplex and further highlight the crucial role of WDR5 in scaffolding the MLL1 core complex.« less

Ligand-Induced Energy Shift and Localization of Kondo Resonances in Cobalt-Based Complexes on Cu(111).

PubMed

Knaak, Thomas; Gruber, Manuel; Lindström, Christoph; Bocquet, Marie-Laure; Heck, Jürgen; Berndt, Richard

2017-11-08

Magnetic sandwich complexes are of particular interest for molecular spintronics. Using scanning tunneling microscopy, we evidence the successful deposition of 1,3,5-tris(η 6 -borabenzene-η 5 -cyclopentadienylcobalt) benzene, a molecule composed of three connected magnetic sandwich units, on Cu(111). Scanning tunneling spectra reveal two distinct spatial-dependent narrow resonances close to the Fermi level for the trimer molecules as well as for molecular fragments composed of one and two magnetic units. With the help of density functional theory, these resonances are interpreted as two Kondo resonances originating from two distinct nondegenerate d-like orbitals. These Kondo resonances are found to have defined spatial extents dictated by the hybridization of the involved orbitals with that of the ligands. These results opens promising perspectives for investigating complex Kondo systems composed of several "Kondo" orbitals.

Complexity Leadership: A Theoretical Perspective

ERIC Educational Resources Information Center

Baltaci, Ali; Balci, Ali

2017-01-01

Complex systems are social networks composed of interactive employees interconnected through collaborative, dynamic ties such as shared goals, perspectives and needs. Complex systems are largely based on "the complex system theory". The complex system theory focuses mainly on finding out and developing strategies and behaviours that…

Visualization of Topology through Simulation

NASA Astrophysics Data System (ADS)

Mulderig, Andrew; Beaucage, Gregory; Vogtt, Karsten; Jiang, Hanqiu

Complex structures can be decomposed into their minimal topological description coupled with complications of tortuosity. We have found that a stick figure representation can account for the topological content of any structure and coupling with scaling measures of tortuosity we can reconstruct an object. This deconstruction is native to static small-angle scattering measurements where we can obtain quantitative measures of the tortuous structure and the minimal topological structure. For example, a crumpled sheet of paper is composed of a minimal sheet structure and parameters reflecting the extent of crumpling. This quantification yields information that can be used to calculate the hydrodynamic radius, radius of gyration, structural conductive pathway, modulus, and other properties of complex structures. The approach is general and has been applied to a wide range of nanostructures from crumpled graphene to branched polymers and unfolded proteins and RNA. In this poster we will demonstrate how simple structural simulations can be used to reconstruct from these parameters a 3d representation of the complex structure through a heuristic approach. Several examples will be given from nano-fractal aggregates.

Sequential and ordered assembly of a large DNA repair complex on undamaged chromatin

PubMed Central

Ziani, Salim; Nagy, Zita; Alekseev, Sergey; Soutoglou, Evi; Egly, Jean-Marc

2014-01-01

In nucleotide excision repair (NER), damage recognition by XPC-hHR23b is described as a critical step in the formation of the preincision complex (PInC) further composed of TFIIH, XPA, RPA, XPG, and ERCC1-XPF. To obtain new molecular insights into the assembly of the PInC, we analyzed its formation independently of DNA damage by using the lactose operator/repressor reporter system. We observed a sequential and ordered self-assembly of the PInC operating upon immobilization of individual NER factors on undamaged chromatin and mimicking that functioning on a bona fide NER substrate. We also revealed that the recruitment of the TFIIH subunit TTDA, involved in trichothiodystrophy group A disorder (TTD-A), was key in the completion of the PInC. TTDA recruits XPA through its first 15 amino acids, depleted in some TTD-A patients. More generally, these results show that proteins forming large nuclear complexes can be recruited sequentially on chromatin in the absence of their natural DNA target and with no reciprocity in their recruitment. PMID:25154395

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

PubMed Central

Hawryluk-Gara, Lisa A.; Platani, Melpomeni; Santarella, Rachel

2008-01-01

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53–Nup155 complex plays a critical role in the processes of NPC and NE assembly. PMID:18256286

Defense Against Pathogens: Structural Insights into the Mechanism of Chitin Induced Activation of Innate Immunity.

PubMed

Squeglia, Flavia; Berisio, Rita; Shibuya, Naoto; Kaku, Hanae

2017-11-24

Pattern recognition receptors on the plant cell surface mediate the recognition of microbe-associated molecular patterns, in a process which activates downstream immune signaling. These receptors are plasma membrane-localized kinases which need to be autophosphorylated to activate downstream responses. Perception of attacks from fungi occurs through recognition of chitin, a polymer of an N-acetylglucosamine which is a characteristic component of the cell walls of fungi. This process is regulated in Arabidopsis by chitin elicitor receptor kinase CERK1. A more complex process characterizes rice, in which regulation of chitin perception is operated by a complex composed of OsCERK1, a homolog of CERK1, and the chitin elicitor binding protein OsCEBiP. Recent literature has provided a mechanistic description of the complex regulation of activation of innate immunity in rice and an advance in the structural description of molecular players involved in this process. This review describes the current status of the understanding of molecular events involved in innate immunity activation in rice. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Design Principle for an Autonomous Post-translational Pattern Formation.

PubMed

Sugai, Shuhei S; Ode, Koji L; Ueda, Hiroki R

2017-04-25

Previous autonomous pattern-formation models often assumed complex molecular and cellular networks. This theoretical study, however, shows that a system composed of one substrate with multisite phosphorylation and a pair of kinase and phosphatase can generate autonomous spatial information, including complex stripe patterns. All (de-)phosphorylation reactions are described with a generic Michaelis-Menten scheme, and all species freely diffuse without pre-existing gradients. Computational simulation upon >23,000,000 randomly generated parameter sets revealed the design motifs of cyclic reaction and enzyme sequestration by slow-diffusing substrates. These motifs constitute short-range positive and long-range negative feedback loops to induce Turing instability. The width and height of spatial patterns can be controlled independently by distinct reaction-diffusion processes. Therefore, multisite reversible post-translational modification can be a ubiquitous source for various patterns without requiring other complex regulations such as autocatalytic regulation of enzymes and is applicable to molecular mechanisms for inducing subcellular localization of proteins driven by post-translational modifications. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

A Refined Model for the TSG-6 Link Module in Complex with Hyaluronan

PubMed Central

Higman, Victoria A.; Briggs, David C.; Mahoney, David J.; Blundell, Charles D.; Sattelle, Benedict M.; Dyer, Douglas P.; Green, Dixy E.; DeAngelis, Paul L.; Almond, Andrew; Milner, Caroline M.; Day, Anthony J.

2014-01-01

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was 13C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a d-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation. PMID:24403066

Network representations of immune system complexity

PubMed Central

Subramanian, Naeha; Torabi-Parizi, Parizad; Gottschalk, Rachel A.; Germain, Ronald N.; Dutta, Bhaskar

2015-01-01

The mammalian immune system is a dynamic multi-scale system composed of a hierarchically organized set of molecular, cellular and organismal networks that act in concert to promote effective host defense. These networks range from those involving gene regulatory and protein-protein interactions underlying intracellular signaling pathways and single cell responses to increasingly complex networks of in vivo cellular interaction, positioning and migration that determine the overall immune response of an organism. Immunity is thus not the product of simple signaling events but rather non-linear behaviors arising from dynamic, feedback-regulated interactions among many components. One of the major goals of systems immunology is to quantitatively measure these complex multi-scale spatial and temporal interactions, permitting development of computational models that can be used to predict responses to perturbation. Recent technological advances permit collection of comprehensive datasets at multiple molecular and cellular levels while advances in network biology support representation of the relationships of components at each level as physical or functional interaction networks. The latter facilitate effective visualization of patterns and recognition of emergent properties arising from the many interactions of genes, molecules, and cells of the immune system. We illustrate the power of integrating ‘omics’ and network modeling approaches for unbiased reconstruction of signaling and transcriptional networks with a focus on applications involving the innate immune system. We further discuss future possibilities for reconstruction of increasingly complex cellular and organism-level networks and development of sophisticated computational tools for prediction of emergent immune behavior arising from the concerted action of these networks. PMID:25625853

K6 linked polyubiquitylation of FADD by CHIP prevents death inducing signaling complex formation suppressing cell death.

PubMed

Seo, Jinho; Lee, Eun-Woo; Shin, Jihye; Seong, Daehyeon; Nam, Young Woo; Jeong, Manhyung; Lee, Seon-Hyeong; Lee, Cheolju; Song, Jaewhan

2018-05-23

Fas-associated death domain (FADD) is an adaptor protein recruiting complexes of caspase 8 to death ligand receptors to induce extrinsic apoptotic cell death in response to a TNF superfamily member. Although, formation of the complex of FADD and caspase 8 upon death stimuli has been studied in detail, posttranslational modifications fine-tuning these processes have yet to be identified. Here we revealed that K6-linked polyubiquitylation of FADD on lysines 149 and 153 mediated by C terminus HSC70-interacting protein (CHIP) plays an important role in preventing formation of the death inducing signaling complex (DISC), thus leading to the suppression of cell death. Cells depleted of CHIP showed higher sensitivity toward death ligands such as FasL and TRAIL, leading to upregulation of DISC formation composed of a death receptor, FADD, and caspase 8. CHIP was able to bind to FADD, induce K6-linked polyubiquitylation of FADD, and suppress DISC formation. By mass spectrometry, lysines 149 and 153 of FADD were found to be responsible for CHIP-mediated FADD ubiquitylation. FADD mutated at these sites was capable of more potent cell death induction as compared with the wild type and was no longer suppressed by CHIP. On the other hand, CHIP deficient in E3 ligase activity was not capable of suppressing FADD function and of FADD ubiquitylation. CHIP depletion in ME-180 cells induced significant sensitization of these cells toward TRAIL in xenograft analyses. These results imply that K6-linked ubiquitylation of FADD by CHIP is a crucial checkpoint in cytokine-dependent extrinsic apoptosis.

Multiple intermediates on the energy landscape of a 15-HEAT-repeat protein

PubMed Central

Tsytlonok, Maksym; Craig, Patricio O.; Sivertsson, Elin; Serquera, David; Perrett, Sarah; Best, Robert B.; Wolynes, Peter G.; Itzhaki, Laura S.

2014-01-01

Repeat proteins are a special class of modular, non-globular proteins composed of small structural motifs arrayed to form elongated architectures and stabilised solely by short-range contacts. We find a remarkable complexity in the unfolding of the large HEAT repeat protein PR65/A. In contrast to what has been seen for small repeat proteins in which unfolding propagates from one end, the HEAT array of PR65/A ruptures at multiple distant sites, leading to intermediate states with non-contiguous folded subdomains. Kinetic analysis allows us to define a network of intermediates and to delineate the pathways that connect them. There is a dominant sequence of unfolding, reflecting a non-uniform distribution of stability across the repeat array; however the unfolding of certain intermediates is competitive, leading to parallel pathways. Theoretical models accounting for the heterogeneous contact density in the folded structure are able to rationalize the variation in stability across the array. This variation in stability also suggests how folding may direct function in a large repeat protein: The stability distribution enables certain regions to present rigid motifs for molecular recognition while affording others flexibility to broaden the search area as in a fly-casting mechanism. Thus PR65/A uses the two ends of the repeat array to bind diverse partners and thereby coordinate the dephosphorylation of many different substrates and of multiple sites within hyperphosphorylated substrates. PMID:24120762

Designed protein reveals structural determinants of extreme kinetic stability

PubMed Central

Broom, Aron; Ma, S. Martha; Xia, Ke; Rafalia, Hitesh; Trainor, Kyle; Colón, Wilfredo; Gosavi, Shachi; Meiering, Elizabeth M.

2015-01-01

The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge. PMID:26554002

Molecular cloning and subcellular localization of Tektin2-binding protein 1 (Ccdc 172) in rat spermatozoa.

PubMed

Yamaguchi, Airi; Kaneko, Takane; Inai, Tetsuichiro; Iida, Hiroshi

2014-04-01

Tektins (TEKTs) are composed of a family of filament-forming proteins localized in cilia and flagella. Five types of mammalian TEKTs have been reported, all of which have been verified to be present in sperm flagella. TEKT2, which is indispensable for sperm structure, mobility, and fertilization, was present at the periphery of the outer dense fiber (ODF) in the sperm flagella. By yeast two-hybrid screening, we intended to isolate flagellar proteins that could interact with TEKT2, which resulted in the isolation of novel two genes from the mouse testis library, referred as a TEKT2-binding protein 1 (TEKT2BP1) and -protein 2 (TEKT2BP2). In this study, we characterized TEKT2BP1, which is registered as a coiled-coil domain-containing protein 172 (Ccdc172) in the latest database. RT-PCR analysis indicated that TEKT2BP1 was predominantly expressed in rat testis and that its expression was increased after 3 weeks of postnatal development. Immunocytochemical studies discovered that TEKT2BP1 localized in the middle piece of rat spermatozoa, predominantly concentrated at the mitochondria sheath of the flagella. We hypothesize that the TEKT2-TEKT2BP1 complex might be involved in the structural linkage between the ODF and mitochondria in the middle piece of the sperm flagella.

Influence of lipids on the interfacial disposition of respiratory syncytical virus matrix protein.

PubMed

McPhee, Helen K; Carlisle, Jennifer L; Beeby, Andrew; Money, Victoria A; Watson, Scott M D; Yeo, R Paul; Sanderson, John M

2011-01-04

The propensity of a matrix protein from an enveloped virus of the Mononegavirales family to associate with lipids representative of the viral envelope has been determined using label-free methods, including tensiometry and Brewster angle microscopy on lipid films at the air-water interface and atomic force microscopy on monolayers transferred to OTS-treated silicon wafers. This has enabled factors that influence the disposition of the protein with respect to the lipid interface to be characterized. In the absence of sphingomyelin, respiratory syncytial virus matrix protein penetrates monolayers composed of mixtures of phosphocholines with phosphoethanolamines or cholesterol at the air-water interface. In ternary mixtures composed of sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol, the protein exhibits two separate behaviors: (1) peripheral association with the surface of sphingomyelin-rich domains and (2) penetration of sphingomyelin-poor domains. Prolonged incubation of the protein with mixtures of phosphocholines and phosphoethanolamines leads to the formation of helical protein assemblies of uniform diameter that demonstrate an inherent propensity of the protein to assemble into a filamentous form.

Structural studies of human glioma pathogenesis-related protein 1

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

Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu; Koski, Raymond A.; Bonafé, Nathalie

2011-10-01

Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structuresmore » of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.« less

Sound Richness of Music Might Be Mediated by Color Perception: A PET Study.

PubMed

Satoh, Masayuki; Nagata, Ken; Tomimoto, Hidekazu

2015-01-01

We investigated the role of the fusiform cortex in music processing with the use of PET, focusing on the perception of sound richness. Musically naïve subjects listened to familiar melodies with three kinds of accompaniments: (i) an accompaniment composed of only three basic chords (chord condition), (ii) a simple accompaniment typically used in traditional music text books in elementary school (simple condition), and (iii) an accompaniment with rich and flowery sounds composed by a professional composer (complex condition). Using a PET subtraction technique, we studied changes in regional cerebral blood flow (rCBF) in simple minus chord, complex minus simple, and complex minus chord conditions. The simple minus chord, complex minus simple, and complex minus chord conditions regularly showed increases in rCBF at the posterior portion of the inferior temporal gyrus, including the LOC and fusiform gyrus. We may conclude that certain association cortices such as the LOC and the fusiform cortex may represent centers of multisensory integration, with foreground and background segregation occurring at the LOC level and the recognition of richness and floweriness of stimuli occurring in the fusiform cortex, both in terms of vision and audition.

The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex.

PubMed

Lindenstrauß, Ute; Skorupa, Philipp; McDowall, Jennifer S; Sargent, Frank; Pinske, Constanze

2017-08-11

The assembly of multi-protein complexes requires the concerted synthesis and maturation of its components and subsequently their co-ordinated interaction. The membrane-bound formate hydrogenlyase (FHL) complex is the primary hydrogen-producing enzyme in Escherichia coli and is composed of seven subunits mostly encoded within the hycA-I operon for [NiFe]-hydrogenase-3 (Hyd-3). The HycH protein is predicted to have an accessory function and is not part of the final structural FHL complex. In this work, a mutant strain devoid of HycH was characterised and found to have significantly reduced FHL activity due to the instability of the electron transfer subunits. HycH was shown to interact specifically with the unprocessed species of HycE, the catalytic hydrogenase subunit of the FHL complex, at different stages during the maturation and assembly of the complex. Variants of HycH were generated with the aim of identifying interacting residues and those that influence activity. The R70/71/K72, the Y79, the E81 and the Y128 variant exchanges interrupt the interaction with HycE without influencing the FHL activity. In contrast, FHL activity, but not the interaction with HycE, was negatively influenced by H37 exchanges with polar residues. Finally, a HycH Y30 variant was unstable. Surprisingly, an overlapping function between HycH with its homologous counterpart HyfJ from the operon encoding [NiFe]-hydrogenase-4 (Hyd-4) was identified and this is the first example of sharing maturation machinery components between Hyd-3 and Hyd-4 complexes. The data presented here show that HycH has a novel dual role as an assembly chaperone for a cytoplasmic [NiFe]-hydrogenase. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Nanofibers formed through pi...pi stacking of the complexes of glucosyl-C2-salicyl-imine and phenylalanine: characterization by microscopy, modeling by molecular mechanics, and interaction by alpha-helical and beta-sheet proteins.

PubMed

Acharya, Amitabha; Ramanujam, Balaji; Mitra, Atanu; Rao, Chebrolu P

2010-07-27

This paper deals with the self-assembly of the 1:1 complex of two different amphiphiles, namely, a glucosyl-salicyl-imino conjugate (L) and phenylalanine (Phe), forming nanofibers over a period of time through pi...pi interactions. Significant enhancement observed in the fluorescence intensity of L at approximately 423 nm band and the significant decrease observed in the absorbance of the approximately 215 nm band are some characteristics of this self-assembly. Matrix-assisted laser desorption ionization/time of flight titration carried out at different time intervals supports the formation of higher aggregates. Atomic force microscopy (AFM), transmission electron microscopy, and scanning electron miscroscopy results showed the formation of nanofibers for the solutions of L with phenylalanine. In dynamic light scattering measurements, the distribution of the particles extends to a higher diameter range over time, indicating a slow kinetic process of assembly. Similar spectral and microscopy studies carried out with the control molecules support the role of the amino acid moiety over the simple -COOH moiety as well as the side chain phenyl moiety in association with the amino acid, in the formation of these fibers. All these observations support the presence of pi...pi interactions between the initially formed 1:1 complexes leading to the fiber formation. The aggregation of 1:1 complexes leading to fibers followed by the formation of bundles has been modeled by molecular mechanics studies. Thus the fiber formation with L is limited to phenylalanine and not to any other naturally occurring amino acid and hence a polymer composed of two different biocompatible amphiphiles. AFM studies carried out between the fiber forming mixture and proteins resulted in the observation that only BSA selectively adheres to the fiber among the three alpha-helical and two beta-sheet proteins studied and hence may be of use in some medical applications.

Isolation, folding and structural investigations of the amino acid transporter OEP16

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

Ni, Da Qun; Zook, James; Klewer, Douglas A.

2011-12-01

Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on amore » Ni-NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of a-helices. 15N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.« less

Isolation, folding and structural investigations of the amino acid transporter OEP16.

PubMed

Ni, Da Qun; Zook, James; Klewer, Douglas A; Nieman, Ronald A; Soll, J; Fromme, Petra

2011-12-01

Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni-NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. ¹⁵N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection. Copyright © 2011. Published by Elsevier Inc.

Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function*

PubMed Central

Lowery, Jason; Kuczmarski, Edward R.; Herrmann, Harald; Goldman, Robert D.

2015-01-01

Intermediate filaments (IFs) are composed of one or more members of a large family of cytoskeletal proteins, whose expression is cell- and tissue type-specific. Their importance in regulating the physiological properties of cells is becoming widely recognized in functions ranging from cell motility to signal transduction. IF proteins assemble into nanoscale biopolymers with unique strain-hardening properties that are related to their roles in regulating the mechanical integrity of cells. Furthermore, mutations in the genes encoding IF proteins cause a wide range of human diseases. Due to the number of different types of IF proteins, we have limited this short review to cover structure and function topics mainly related to the simpler homopolymeric IF networks composed of vimentin, and specifically for diseases, the related muscle-specific desmin IF networks. PMID:25957409

A bone substitute with high affinity for vitamin D-binding protein―relationship with niche of osteoclasts

PubMed Central

Ikeda, Tohru; Kasai, Michiyuki; Tatsukawa, Eri; Kamitakahara, Masanobu; Shibata, Yasuaki; Yokoi, Taishi; Nemoto, Takayuki K; Ioku, Koji

2014-01-01

The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod-shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular-shaped particles (CHA). The two ceramics adsorbed serum albumin and γ-globulin to similar extents, but affinity for γ-globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D-binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8-week-old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP-macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod-shaped particles influences its potent osteoclast homing activity and local bone metabolism. PMID:24286277

α-dystroglycan is a potential target of matrix metalloproteinase MMP-2.

PubMed

Sbardella, Diego; Sciandra, Francesca; Gioia, Magda; Marini, Stefano; Gori, Alessandro; Giardina, Bruno; Tarantino, Umberto; Coletta, Massimo; Brancaccio, Andrea; Bozzi, Manuela

2015-01-01

Dystroglycan (DG) is a member of the glycoprotein complex associated to dystrophin and composed by two subunits, the β-DG, a transmembrane protein, and the α-DG, an extensively glycosylated extracellular protein. The β-DG ectodomain degradation by the matrix metallo-proteinases (i.e., MMP-2 and MMP-9) in both, pathological and physiological conditions, has been characterized in detail in previous publications. Since the amounts of α-DG and β-DG at the cell surface decrease when gelatinases are up-regulated, we investigated the degradation of α-DG subunit by MMP-2. Present data show, for the first time, that the proteolysis of α-DG indeed occurs on a native glycosylated molecule enriched from rabbit skeletal muscle. In order to characterize the α-DG portion, which is more prone to cleavage by MMP-2, we performed different degradations on tailored recombinant domains of α-DG spanning the whole subunit. The overall bulk of results casts light on a relevant susceptibility of the α-DG to MMP-2 degradation with particular reference to its C-terminal domain, thus opening a new scenario on the role of gelatinases (in particular of MMP-2) in the degradation of this glycoprotein complex, taking place in the course of pathological processes. Copyright © 2014. Published by Elsevier B.V.

The mitochondrial electron transfer flavoprotein complex is essential for survival of Arabidopsis in extended darkness.

PubMed

Ishizaki, Kimitsune; Schauer, Nicolas; Larson, Tony R; Graham, Ian A; Fernie, Alisdair R; Leaver, Christopher J

2006-09-01

In mammals, the electron transfer flavoprotein (ETF) is a heterodimeric protein composed of two subunits, alpha and beta, that is responsible for the oxidation of at least nine mitochondrial matrix flavoprotein dehydrogenases. Electrons accepted by ETF are further transferred to the main respiratory chain via the ETF ubiquinone oxide reductase (ETFQO). Sequence analysis of the unique Arabidopsis homologues of two subunits of ETF revealed their high similarity to both subunits of the mammalian ETF. Yeast two-hybrid experiments showed that the Arabidopsis ETFalpha and ETFbeta can form a heteromeric protein. Isolation and characterization of two independent T-DNA insertional Arabidopsis mutants of the ETFbeta gene revealed accelerated senescence and early death compared to wild-type during extended darkness. Furthermore in contrast to wild-type, the etfb mutants demonstrated a significant accumulation of several amino acids, isovaleryl CoA and phytanoyl CoA during dark-induced carbohydrate deprivation. These phenotypic characteristics of etfb mutants are broadly similar to those that we observed previously in Arabidopsis etfqo mutants, suggesting functional association between ETF and ETFQO in Arabidopsis, and confirming the essential roles of the ETF/ETFQO electron transfer complex in the catabolism of leucine and involvement in the chlorophyll degradation pathway activated during dark-induced carbohydrate deprivation.

The nuclear membrane-associated honeycomb structure of the unicellular organism Amoeba proteus: on the search for homologies with the nuclear lamina of metazoa.

PubMed

Schmidt, M; Grossmann, U; Krohne, G

1995-07-01

In the protozoon Amoeba proteus, a complex and highly organized structure with the morphology of a honeycomb is associated with the nucleoplasmic surface of the nuclear membrane. We have tested whether this structure exhibits similarity to the nuclear lamina of metazoic organisms. First, we have shown that the honeycomb layer is composed of 3 to 5 nm thick protein fibrils resistant to treatment with detergent, high salt, and digestion with nucleases, thus possessing properties typical for karyoskeletal elements. However, in contrast to the meshwork of lamin filaments in somatic cells of metazoic organisms, the honeycomb layer is not tightly anchored to the nucleoplasmic side of pore complexes, or to the inner nuclear membrane. Second, in microinjection experiments we investigated whether fluorescently labeled lamins of Xenopus laevis (lamins A and LI) and Drosophila melanogaster (lamin Dmo) were able to associate in vivo with the Amoeba proteus honeycomb structure. In microinjected amoeba these three lamins were efficiently transported into the nucleus, but did not associate with the nuclear envelope. Our results suggest that the Amoeba proteus nuclear envelope, including the honeycomb layer, does not contain proteins exhibiting high homologies to lamins of metazoan species thus preventing the localized assembly of microinjected lamins along the nuclear periphery.

Characterization of C-ring component assembly in flagellar motors from amino acid coevolution

PubMed Central

dos Santos, Ricardo Nascimento; Khan, Shahid

2018-01-01

Bacterial flagellar motility, an important virulence factor, is energized by a rotary motor localized within the flagellar basal body. The rotor module consists of a large framework (the C-ring), composed of the FliG, FliM and FliN proteins. FliN and FliM contacts the FliG torque ring to control the direction of flagellar rotation. We report that structure-based models constrained only by residue coevolution can recover the binding interface of atomic X-ray dimer complexes with remarkable accuracy (approx. 1 Å RMSD). We propose a model for FliM–FliN heterodimerization, which agrees accurately with homologous interfaces as well as in situ cross-linking experiments, and hence supports a proposed architecture for the lower portion of the C-ring. Furthermore, this approach allowed the identification of two discrete and interchangeable homodimerization interfaces between FliM middle domains that agree with experimental measurements and might be associated with C-ring directional switching dynamics triggered upon binding of CheY signal protein. Our findings provide structural details of complex formation at the C-ring that have been difficult to obtain with previous methodologies and clarify the architectural principle that underpins the ultra-sensitive allostery exhibited by this ring assembly that controls the clockwise or counterclockwise rotation of flagella. PMID:29892378

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

Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji

Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a verymore » low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V{alpha}/V{beta} region of the TCR. These findings provide new insights into the binding of sTCRs to p/MHCs and will hopefully be instrumental in establishing functional sTCR as a diagnostic and therapeutic tool for cancer.« less

Chemical annotation of small and peptide-like molecules at the Protein Data Bank

PubMed Central

Young, Jasmine Y.; Feng, Zukang; Dimitropoulos, Dimitris; Sala, Raul; Westbrook, John; Zhuravleva, Marina; Shao, Chenghua; Quesada, Martha; Peisach, Ezra; Berman, Helen M.

2013-01-01

Over the past decade, the number of polymers and their complexes with small molecules in the Protein Data Bank archive (PDB) has continued to increase significantly. To support scientific advancements and ensure the best quality and completeness of the data files over the next 10 years and beyond, the Worldwide PDB partnership that manages the PDB archive is developing a new deposition and annotation system. This system focuses on efficient data capture across all supported experimental methods. The new deposition and annotation system is composed of four major modules that together support all of the processing requirements for a PDB entry. In this article, we describe one such module called the Chemical Component Annotation Tool. This tool uses information from both the Chemical Component Dictionary and Biologically Interesting molecule Reference Dictionary to aid in annotation. Benchmark studies have shown that the Chemical Component Annotation Tool provides significant improvements in processing efficiency and data quality. Database URL: http://wwpdb.org PMID:24291661

Cracking the ANP32 whips: Important functions, unequal requirement, and hints at disease implications

PubMed Central

Reilly, Patrick T; Yu, Yun; Hamiche, Ali; Wang, Lishun

2014-01-01

The acidic (leucine-rich) nuclear phosphoprotein 32 kDa (ANP32) family is composed of small, evolutionarily conserved proteins characterized by an N-terminal leucine-rich repeat domain and a C-terminal low-complexity acidic region. The mammalian family members (ANP32A, ANP32B, and ANP32E) are ascribed physiologically diverse functions including chromatin modification and remodelling, apoptotic caspase modulation, protein phosphatase inhibition, as well as regulation of intracellular transport. In addition to reviewing the widespread literature on the topic, we present a concept of the ANP32s as having a whip-like structure. We also present hypotheses that ANP32C and other intronless sequences should not currently be considered bona fide family members, that their disparate necessity in development may be due to compensatory mechanisms, that their contrasting roles in cancer are likely context-dependent, along with an underlying hypothesis that ANP32s represent an important node of physiological regulation by virtue of their diverse biochemical activities. PMID:25156960

High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions

PubMed Central

Nahmani, Marc; Lanahan, Conor; DeRosier, David; Turrigiano, Gina G.

2017-01-01

Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated localization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets. PMID:28348224

Structure and membrane remodeling activity of ESCRT-III helical polymers

PubMed Central

McCullough, John; Clippinger, Amy K.; Talledge, Nathaniel; Skowyra, Michael L.; Saunders, Marissa G.; Naismith, Teresa V.; Colf, Leremy A.; Afonine, Pavel; Arthur, Christopher; Sundquist, Wesley I.; Hanson, Phyllis I.; Frost, Adam

2015-01-01

The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 Å resolution cryo-EM reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, CHMP1B and IST1. The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecture, and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively-curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature. PMID:26634441

RISC-interacting clearing 3'- 5' exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana.

PubMed

Zhang, Zhonghui; Hu, Fuqu; Sung, Min Woo; Shu, Chang; Castillo-González, Claudia; Koiwa, Hisashi; Tang, Guiliang; Dickman, Martin; Li, Pingwei; Zhang, Xiuren

2017-05-02

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5' products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC.

Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

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

Schlunzen, Frank; Zarivach, Raz; Harms, Jörg

2009-10-07

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavitymore » and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.« less

Chemical Synthesis of Oligosaccharides Related to the Cell Walls of Plants and Algae.

PubMed

Kinnaert, Christine; Daugaard, Mathilde; Nami, Faranak; Clausen, Mads H

2017-09-13

Plant cell walls are composed of an intricate network of polysaccharides and proteins that varies during the developmental stages of the cell. This makes it very challenging to address the functions of individual wall components in cells, especially for highly complex glycans. Fortunately, structurally defined oligosaccharides can be used as models for the glycans, to study processes such as cell wall biosynthesis, polysaccharide deposition, protein-carbohydrate interactions, and cell-cell adhesion. Synthetic chemists have focused on preparing such model compounds, as they can be produced in good quantities and with high purity. This Review contains an overview of those plant and algal polysaccharides that have been elucidated to date. The majority of the content is devoted to detailed summaries of the chemical syntheses of oligosaccharide fragments of cellulose, hemicellulose, pectin, and arabinogalactans, as well as glycans unique to algae. Representative synthetic routes within each class are discussed in detail, and the progress in carbohydrate chemistry over recent decades is highlighted.

Chemical annotation of small and peptide-like molecules at the Protein Data Bank.

PubMed

Young, Jasmine Y; Feng, Zukang; Dimitropoulos, Dimitris; Sala, Raul; Westbrook, John; Zhuravleva, Marina; Shao, Chenghua; Quesada, Martha; Peisach, Ezra; Berman, Helen M

2013-01-01

Over the past decade, the number of polymers and their complexes with small molecules in the Protein Data Bank archive (PDB) has continued to increase significantly. To support scientific advancements and ensure the best quality and completeness of the data files over the next 10 years and beyond, the Worldwide PDB partnership that manages the PDB archive is developing a new deposition and annotation system. This system focuses on efficient data capture across all supported experimental methods. The new deposition and annotation system is composed of four major modules that together support all of the processing requirements for a PDB entry. In this article, we describe one such module called the Chemical Component Annotation Tool. This tool uses information from both the Chemical Component Dictionary and Biologically Interesting molecule Reference Dictionary to aid in annotation. Benchmark studies have shown that the Chemical Component Annotation Tool provides significant improvements in processing efficiency and data quality. Database URL: http://wwpdb.org.

Novel mutations in the CDKL5 gene in complex genotypes associated with West syndrome with variable phenotype: First description of somatic mosaic state.

PubMed

Jdila, Marwa Ben; Issa, Abir Ben; Khabou, Boudour; Rhouma, Bochra Ben; Kamoun, Fatma; Ammar-Keskes, Leila; Triki, Chahnez; Fakhfakh, Faiza

2017-10-01

West syndrome is a rare epileptic encephalopathy of early infancy, characterized by epileptic spasms, hypsarrhythmia, and psychomotor retardation beginning in the first year of life. The present study reports the clinical, molecular and bioinformatic investigation in the three studied West patients. The results revealed a complex genotype with more than one mutation in each patient including the known mutations c.1910C>G (P2, P3); c.2372A>C in P3 and c.2395C>G in P1 and novel variants including c.616G>A, shared by the three patients P1, P2 and P3; c.1403G>C shared by P2 and P3 and c.2288A>G in patient P1. All the mutations were at somatic mosaic state and were de novo in the patients except ones (c.2372A>C). To our knowledge; the somatic mosaic state is described for the first time in patients with West syndrome. Five identified mutations were located in the C-terminal domain of the protein, while the novel mutation (c.616G>A) was in the catalytic domain. Bioinformatic tools predicted that this latter is the most pathogenic substitution affecting 3D protein structure and the secondary mRNA structure. Complex genotype composed of different combinations of mutations in each patient seems to be related to the phenotype variability. Copyright © 2017. Published by Elsevier B.V.

Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei

PubMed Central

Kamina, Anyango D.; Jaremko, Daniel; Christen, Linda

2017-01-01

ABSTRACT Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei, the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T. brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei. IMPORTANCE Trypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the regulatory checkpoints during ribosome biogenesis. We have previously characterized the 5S RNP in T. brucei and showed that trypanosome-specific proteins P34 and P37 are part of this complex. In this study, we characterize for the first time the interactions of the homolog of the assembly factor Rpf2 with members of the 5S RNP in another organism besides fungi. Our studies show that Rpf2 is essential in T. brucei and that it forms unique interactions within the 5S RNP, particularly with P34 and P37. These studies have identified parasite-specific interactions that can potentially function as new therapeutic targets against sleeping sickness. PMID:29062898

Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei.

PubMed

Kamina, Anyango D; Jaremko, Daniel; Christen, Linda; Williams, Noreen

2017-01-01

Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei , the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T . brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei . IMPORTANCE Trypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the regulatory checkpoints during ribosome biogenesis. We have previously characterized the 5S RNP in T. brucei and showed that trypanosome-specific proteins P34 and P37 are part of this complex. In this study, we characterize for the first time the interactions of the homolog of the assembly factor Rpf2 with members of the 5S RNP in another organism besides fungi. Our studies show that Rpf2 is essential in T. brucei and that it forms unique interactions within the 5S RNP, particularly with P34 and P37. These studies have identified parasite-specific interactions that can potentially function as new therapeutic targets against sleeping sickness.

Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

PubMed Central

Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

2012-01-01

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

Folding and unfolding single RNA molecules under tension

PubMed Central

Woodside, Michael T; García-García, Cuauhtémoc; Block, Steven M

2010-01-01

Single-molecule force spectroscopy constitutes a powerful method for probing RNA folding: it allows the kinetic, energetic, and structural properties of intermediate and transition states to be determined quantitatively, yielding new insights into folding pathways and energy landscapes. Recent advances in experimental and theoretical methods, including fluctuation theorems, kinetic theories, novel force clamps, and ultrastable instruments, have opened new avenues for study. These tools have been used to probe folding in simple model systems, for example, RNA and DNA hairpins. Knowledge gained from such systems is helping to build our understanding of more complex RNA structures composed of multiple elements, as well as how nucleic acids interact with proteins involved in key cellular activities, such as transcription and translation. PMID:18786653

Silk-regulated hierarchical hollow magnetite/carbon nanocomposite spheroids for lithium-ion battery anodes.

PubMed

Sheng, Weiqin; Zhu, Guobin; Kaplan, David L; Cao, Chuanbao; Zhu, Hesun; Lu, Qiang

2015-03-20

Hierarchical olive-like structured carbon-Fe3O4 nanocomposite particles composed of a hollow interior and a carbon coated surface are prepared by a facile, silk protein-assisted hydrothermal method. Silk nanofibers as templates and carbon precursors first regulate the formation of hollow Fe2O3 microspheres and then they are converted into carbon by a reduction process into Fe3O4. This process significantly simplifies the fabrication and carbon coating processes to form complex hollow structures. When tested as anode materials for lithium-ion batteries, these hollow carbon-coated particles exhibit high capacity (900 mAh g(-1)), excellent cycle stability (180 cycles) and rate performance due to their unique hierarchical hollow structure and carbon coating.

Pretargeting immunotherapy: a novel treatment approach for systemic amyloidosis.

PubMed

Wall, Jonathan S; Foster, James S; Martin, Emily B; Kennel, Stephen J

2017-09-01

The amyloidoses are a complex group of disorders characterized by the deposition of proteinaceous amyloid fibrils in vital organs. The deposits are nonimmunogenic and may be composed of one of more than 35 proteins. We have developed a two-stage immunotherapeutic approach using peptides that recognize most, if not all, amyloid deposits to facilitate amyloid clearance. In the first embodiment, we have developed a bifunctional peptope to enhance and expand the utility of currently available antibodies. In the second, we have generated peptide-reactive antibodies that can be targeted to the amyloid deposits by peptides thereby providing alternative reagents for immunotherapy of amyloidosis. These technologies provide tools for treating the many forms of amyloid disease, restoring organ function and enhancing patient survival.

Depletion of Plasmodium berghei plasmoredoxin reveals a non-essential role for life cycle progression of the malaria parasite.

PubMed

Buchholz, Kathrin; Rahlfs, Stefan; Schirmer, R Heiner; Becker, Katja; Matuschewski, Kai

2008-06-25

Proliferation of the pathogenic Plasmodium asexual blood stages in host erythrocytes requires an exquisite capacity to protect the malaria parasite against oxidative stress. This function is achieved by a complex antioxidant defence system composed of redox-active proteins and low MW antioxidants. Here, we disrupted the P. berghei plasmoredoxin gene that encodes a parasite-specific 22 kDa member of the thioredoxin superfamily. The successful generation of plasmoredoxin knockout mutants in the rodent model malaria parasite and phenotypic analysis during life cycle progression revealed a non-vital role in vivo. Our findings suggest that plasmoredoxin fulfils a specialized and dispensable role for Plasmodium and highlights the need for target validation to inform drug development strategies.

The RNA-binding Protein TDP-43 Selectively Disrupts MicroRNA-1/206 Incorporation into the RNA-induced Silencing Complex*♦

PubMed Central

King, Isabelle N.; Yartseva, Valeria; Salas, Donaldo; Kumar, Abhishek; Heidersbach, Amy; Ando, D. Michael; Stallings, Nancy R.; Elliott, Jeffrey L.; Srivastava, Deepak; Ivey, Kathryn N.

2014-01-01

MicroRNA (miRNA) maturation is regulated by interaction of particular miRNA precursors with specific RNA-binding proteins. Following their biogenesis, mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) where they interact with mRNAs to negatively regulate protein production. However, little is known about how mature miRNAs are regulated at the level of their activity. To address this, we screened for proteins differentially bound to the mature form of the miR-1 or miR-133 miRNA families. These muscle-enriched, co-transcribed miRNA pairs cooperate to suppress smooth muscle gene expression in the heart. However, they also have opposing roles, with the miR-1 family, composed of miR-1 and miR-206, promoting myogenic differentiation, whereas miR-133 maintains the progenitor state. Here, we describe a physical interaction between TDP-43, an RNA-binding protein that forms aggregates in the neuromuscular disease, amyotrophic lateral sclerosis, and the miR-1, but not miR-133, family. Deficiency of the TDP-43 Drosophila ortholog enhanced dmiR-1 activity in vivo. In mammalian cells, TDP-43 limited the activity of both miR-1 and miR-206, but not the miR-133 family, by disrupting their RISC association. Consistent with TDP-43 dampening miR-1/206 activity, protein levels of the miR-1/206 targets, IGF-1 and HDAC4, were elevated in TDP-43 transgenic mouse muscle. This occurred without corresponding Igf-1 or Hdac4 mRNA increases and despite higher miR-1 and miR-206 expression. Our findings reveal that TDP-43 negatively regulates the activity of the miR-1 family of miRNAs by limiting their bioavailability for RISC loading and suggest a processing-independent mechanism for differential regulation of miRNA activity. PMID:24719334

Plasma and Plasma Protein Product Transfusion: A Canadian Blood Services Centre for Innovation Symposium.

PubMed

Zeller, Michelle P; Al-Habsi, Khalid S; Golder, Mia; Walsh, Geraldine M; Sheffield, William P

2015-07-01

Plasma obtained via whole blood donation processing or via apheresis technology can either be transfused directly to patients or pooled and fractionated into plasma protein products that are concentrates of 1 or more purified plasma protein. The evidence base supporting clinical efficacy in most of the indications for which plasma is transfused is weak, whereas high-quality evidence supports the efficacy of plasma protein products in at least some of the clinical settings in which they are used. Transfusable plasma utilization remains composed in part of applications that fall outside of clinical practice guidelines. Plasma contains all of the soluble coagulation factors and is frequently transfused in efforts to restore or reinforce patient hemostasis. The biochemical complexities of coagulation have in recent years been rationalized in newer cell-based models that supplement the cascade hypothesis. Efforts to normalize widely used clinical hemostasis screening test values by plasma transfusion are thought to be misplaced, but superior rapid tests have been slow to emerge. The advent of non-vitamin K-dependent oral anticoagulants has brought new challenges to clinical laboratories in plasma testing and to clinicians needing to reverse non-vitamin K-dependent oral anticoagulants urgently. Current plasma-related controversies include prophylactic plasma transfusion before invasive procedures, plasma vs prothrombin complex concentrates for urgent warfarin reversal, and the utility of increased ratios of plasma to red blood cell units transfused in massive transfusion protocols. The first recombinant plasma protein products to reach the clinic were recombinant hemophilia treatment products, and these donor-free equivalents to factors VIII and IX are now being supplemented with novel products whose circulatory half-lives have been increased by chemical modification or genetic fusion. Achieving optimal plasma utilization is an ongoing challenge in the interconnected worlds of transfusable plasma, plasma protein products, and recombinant and engineered replacements. Copyright © 2015 Elsevier Inc. All rights reserved.

The RNA-binding protein TDP-43 selectively disrupts microRNA-1/206 incorporation into the RNA-induced silencing complex.

PubMed

King, Isabelle N; Yartseva, Valeria; Salas, Donaldo; Kumar, Abhishek; Heidersbach, Amy; Ando, D Michael; Stallings, Nancy R; Elliott, Jeffrey L; Srivastava, Deepak; Ivey, Kathryn N

2014-05-16

MicroRNA (miRNA) maturation is regulated by interaction of particular miRNA precursors with specific RNA-binding proteins. Following their biogenesis, mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) where they interact with mRNAs to negatively regulate protein production. However, little is known about how mature miRNAs are regulated at the level of their activity. To address this, we screened for proteins differentially bound to the mature form of the miR-1 or miR-133 miRNA families. These muscle-enriched, co-transcribed miRNA pairs cooperate to suppress smooth muscle gene expression in the heart. However, they also have opposing roles, with the miR-1 family, composed of miR-1 and miR-206, promoting myogenic differentiation, whereas miR-133 maintains the progenitor state. Here, we describe a physical interaction between TDP-43, an RNA-binding protein that forms aggregates in the neuromuscular disease, amyotrophic lateral sclerosis, and the miR-1, but not miR-133, family. Deficiency of the TDP-43 Drosophila ortholog enhanced dmiR-1 activity in vivo. In mammalian cells, TDP-43 limited the activity of both miR-1 and miR-206, but not the miR-133 family, by disrupting their RISC association. Consistent with TDP-43 dampening miR-1/206 activity, protein levels of the miR-1/206 targets, IGF-1 and HDAC4, were elevated in TDP-43 transgenic mouse muscle. This occurred without corresponding Igf-1 or Hdac4 mRNA increases and despite higher miR-1 and miR-206 expression. Our findings reveal that TDP-43 negatively regulates the activity of the miR-1 family of miRNAs by limiting their bioavailability for RISC loading and suggest a processing-independent mechanism for differential regulation of miRNA activity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cottonseed meal with high level of (+)-Gossypol as diet for broilers

USDA-ARS?s Scientific Manuscript database

Cottonseed is composed of ~22.5% of a high quality protein. This represents 3.5 MMT of protein that could be available for human consumption. Cottonseed protein currently is underutilized because of the presence of a toxic compound called gossypol. Gossypol is biosynthesized by the free radical co...

Design of an efficient medium for heterologous protein production in Yarrowia lipolytica: case of human interferon alpha 2b.

PubMed

Gasmi, Najla; Ayed, Atef; Nicaud, Jean-Marc; Kallel, Héla

2011-05-20

The non conventional yeast Yarrowia lipolytica has aroused a strong industrial interest for heterologous protein production. However most of the studies describing recombinant protein production by this yeast rely on the use of complex media, such media are not convenient for large scale production particularly for products intended for pharmaceutical applications. In addition medium composition can also affect the production yield. Hence it is necessary to design an efficient medium for therapeutic protein expression by this host. Five different media, including four minimal media and a complex medium, were assessed in shake flasks for the production of human interferon alpha 2b (hIFN α2b) by Y. lipolytica under the control of POX2 promoter inducible with oleic acid. The chemically defined medium SM4 formulated by Invitrogen for Pichia pastoris growth was the most suitable. Using statistical experimental design this medium was further optimized. The selected minimal medium consisting in SM4 supplemented with 10 mg/l FeCl₃, 1 g/l glutamate, 5 ml/l PTM1 (Pichia Trace Metals) solution and a vitamin solution composed of myo-inositol, thiamin and biotin was called GNY medium. Compared to shake flask, bioreactor culture in GNY medium resulted in 416-fold increase of hIFN α2b production and 2-fold increase of the biological activity. Furthermore, SM4 enrichment with 5 ml/l PTM1 solution contributed to protect hIFN α2b against the degradation by the 28 kDa protease identified by zymography gel in culture supernatant. The screening of the inhibitory effect of the trace elements present in PTM1 solution on the activity of this protease was achieved using a Box-Behnken design. Statistical data analysis showed that FeCl₃ and MnSO₄ had the most inhibitory effect. We have designed an efficient medium for large scale production of heterologous proteins by Y. lipolytica. The optimized medium GNY is suitable for the production of hIFN α2b with the advantage that no complex nitrogen sources with non-defined composition were required.

Freezing-induced phase separation and spatial microheterogeneity in protein solutions.

PubMed

Dong, Jinping; Hubel, Allison; Bischof, John C; Aksan, Alptekin

2009-07-30

Amid decades of research, the basic mechanisms of lyo-/cryostabilization of proteins and more complex organisms have not yet been fully established. One major bottleneck is the inability to probe into and control the molecular level interactions. The molecular interactions are responsible for the significant differences in the outcome of the preservation processes. (1) In this communication, we have utilized confocal Raman microspectroscopy to quantify the freezing-induced microheterogeneity and phase separation (solid and liquid) in a frozen solution composed of a model protein (lysozyme) and a lyo-/cryoprotectant (trehalose), which experienced different degrees of supercooling. Detailed quantitative spectral analysis was performed across the ice, the freeze-concentrated liquid (FCL) phases, and the interface region between them. It was established that the characteristics of the microstructures observed after freezing depended not only on the concentration of trehalose in the solution but also on the degree of supercooling. It was shown that, when samples were frozen after high supercooling, small amounts of lysozyme and trehalose were occluded in the ice phase. Lysozyme preserved its native-like secondary structure in the FCL region but was denatured in the ice phase. Also, it was observed that induction of freezing after a high degree of supercooling of high trehalose concentrations resulted in aggregation of the sugar and the protein.

SusG: A Unique Cell-Membrane-Associated [alpha]-Amylase from a Prominent Human Gut Symbiont Targets Complex Starch Molecules

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

Koropatkin, Nicole M.; Smith, Thomas J.

SusG is an {alpha}-amylase and part of a large protein complex on the outer surface of the bacterial cell and plays a major role in carbohydrate acquisition by the animal gut microbiota. Presented here, the atomic structure of SusG has an unusual extended, bilobed structure composed of amylase at one end and an unprecedented internal carbohydrate-binding motif at the other. Structural studies further demonstrate that the carbohydrate-binding motif binds maltooligosaccharide distal to, and on the opposite side of, the amylase catalytic site. SusG has an additional starch-binding site on the amylase domain immediately adjacent to the active cleft. Mutagenesis analysismore » demonstrates that these two additional starch-binding sites appear to play a role in catabolism of insoluble starch. However, elimination of these sites has only a limited effect, suggesting that they may have a more important role in product exchange with other Sus components.« less

Formulation and stabilization of nano-/microdispersion systems using naturally occurring edible polyelectrolytes by electrostatic deposition and complexation.

PubMed

Kuroiwa, Takashi; Kobayashi, Isao; Chuah, Ai Mey; Nakajima, Mitsutoshi; Ichikawa, Sosaku

2015-12-01

This review paper presents an overview of the formulation and functionalization of nano-/microdispersion systems composed of edible materials. We first summarized general aspects on the stability of colloidal systems and the roles of natural polyelectrolytes such as proteins and ionic polysaccharides for the formation and stabilization of colloidal systems. Then we introduced our research topics on (1) stabilization of emulsions by the electrostatic deposition using natural polyelectrolytes and (2) formulation of stable nanodispersion systems by complexation of natural polyelectrolytes. In both cases, the preparation procedures were relatively simple, without high energy input or harmful chemical addition. The properties of the nano-/microdispersion systems, such as particle size, surface charge and dispersion stability were significantly affected by the concerned materials and preparation conditions, including the type and concentration of used natural polyelectrolytes. These dispersion systems would be useful for developing novel foods having high functionality and good stability. Copyright © 2015 Elsevier B.V. All rights reserved.

Functional Anatomy of the Human Microprocessor.

PubMed

Nguyen, Tuan Anh; Jo, Myung Hyun; Choi, Yeon-Gil; Park, Joha; Kwon, S Chul; Hohng, Sungchul; Kim, V Narry; Woo, Jae-Sung

2015-06-04

MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an ∼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a "ruler" by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing. Copyright © 2015 Elsevier Inc. All rights reserved.

The Yeast Nuclear Pore Complex and Transport Through It

PubMed Central

Aitchison, John D.; Rout, Michael P.

2012-01-01

Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell’s genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or “Nups”), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC’s role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins. PMID:22419078

Dynamic contact angle analysis of protein adsorption on polysaccharide multilayer's films for biomaterial reendothelialization.

PubMed

Benni, Safiya; Avramoglou, Thierry; Hlawaty, Hanna; Mora, Laurence

2014-01-01

Atherosclerosis is a major cardiovascular disease. One of the side effects is restenosis. The aim of this work was to study the coating of stents by dextran derivates based polyelectrolyte's multilayer (PEM) films in order to increase endothelialization of injured arterial wall after stent implantation. Films were composed with diethylaminoethyl dextran (DEAE) as polycation and dextran sulphate (DS) as polyanion. One film was composed with 4 bilayers of (DEAE-DS)4 and was labeled D-. The other film was the same as D- but with an added terminal layer of DEAE polycation: (DEAE-DS)4-DEAE (labeled D+). The dynamic adsorption/desorption of proteins on the films were characterized by dynamic contact angle (DCA) and atomic force microscopy (AFM). Human endothelial cell (HUVEC) adhesion and proliferation were quantified and correlated to protein adsorption analyzed by DCA for fibronectin, vitronectin, and bovine serum albumin (BSA). Our results showed that the endothelial cell response was optimal for films composed of DS as external layer. Fibronectin was found to be the only protein to exhibit a reversible change in conformation after desorption test. This behavior was only observed for (DEAE-DS)4 films. (DEAE-DS)4 films could enhance HUVEC proliferation in agreement with fibronectin ability to easily change from conformation.

Dynamic Contact Angle Analysis of Protein Adsorption on Polysaccharide Multilayer's Films for Biomaterial Reendothelialization

PubMed Central

Benni, Safiya; Mora, Laurence

2014-01-01

Atherosclerosis is a major cardiovascular disease. One of the side effects is restenosis. The aim of this work was to study the coating of stents by dextran derivates based polyelectrolyte's multilayer (PEM) films in order to increase endothelialization of injured arterial wall after stent implantation. Films were composed with diethylaminoethyl dextran (DEAE) as polycation and dextran sulphate (DS) as polyanion. One film was composed with 4 bilayers of (DEAE-DS)4 and was labeled D−. The other film was the same as D− but with an added terminal layer of DEAE polycation: (DEAE-DS)4-DEAE (labeled D+). The dynamic adsorption/desorption of proteins on the films were characterized by dynamic contact angle (DCA) and atomic force microscopy (AFM). Human endothelial cell (HUVEC) adhesion and proliferation were quantified and correlated to protein adsorption analyzed by DCA for fibronectin, vitronectin, and bovine serum albumin (BSA). Our results showed that the endothelial cell response was optimal for films composed of DS as external layer. Fibronectin was found to be the only protein to exhibit a reversible change in conformation after desorption test. This behavior was only observed for (DEAE-DS)4 films. (DEAE-DS)4 films could enhance HUVEC proliferation in agreement with fibronectin ability to easily change from conformation. PMID:25276808

Molecular basis for the interaction between stress-inducible phosphoprotein 1 (STIP1) and S100A1.

PubMed

Maciejewski, Andrzej; Prado, Vania F; Prado, Marco A M; Choy, Wing-Yiu

2017-05-16

Stress-inducible phosphoprotein 1 (STIP1) is a cellular co-chaperone, which regulates heat-shock protein 70 (Hsp70) and Hsp90 activity during client protein folding. Members of the S100 family of dimeric calcium-binding proteins have been found to inhibit Hsp association with STIP1 through binding of STIP1 tetratricopeptide repeat (TPR) domains, possibly regulating the chaperone cycle. Here, we investigated the molecular basis of S100A1 binding to STIP1. We show that three S100A1 dimers associate with one molecule of STIP1 in a calcium-dependent manner. Isothermal titration calorimetry revealed that individual STIP1 TPR domains, TPR1, TPR2A and TPR2B, bind a single S100A1 dimer with significantly different affinities and that the TPR2B domain possesses the highest affinity for S100A1. S100A1 bound each TPR domain through a common binding interface composed of α-helices III and IV of each S100A1 subunit, which is only accessible following a large conformational change in S100A1 upon calcium binding. The TPR2B-binding site for S100A1 was predominately mapped to the C-terminal α-helix of TPR2B, where it is inserted into the hydrophobic cleft of an S100A1 dimer, suggesting a novel binding mechanism. Our data present the structural basis behind STIP1 and S100A1 complex formation, and provide novel insights into TPR module-containing proteins and S100 family member complexes. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Characterization of the influence of mediator complex in HIV-1 transcription.

PubMed

Ruiz, Alba; Pauls, Eduardo; Badia, Roger; Riveira-Muñoz, Eva; Clotet, Bonaventura; Ballana, Ester; Esté, José A

2014-10-03

HIV-1 exploits multiple host proteins during infection. siRNA-based screenings have identified new proteins implicated in different pathways of the viral cycle that participate in a broad range of cellular functions. The human Mediator complex (MED) is composed of 28 elements and represents a fundamental component of the transcription machinery, interacting with the RNA polymerase II enzyme and regulating its ability to express genes. Here, we provide an evaluation of the MED activity on HIV replication. Knockdown of 9 out of 28 human MED proteins significantly impaired viral replication without affecting cell viability, including MED6, MED7, MED11, MED14, MED21, MED26, MED27, MED28, and MED30. Impairment of viral replication by MED subunits was at a post-integration step. Inhibition of early HIV transcripts was observed by siRNA-mediated knockdown of MED6, MED7, MED11, MED14, and MED28, specifically affecting the transcription of the nascent viral mRNA transactivation-responsive element. In addition, MED14 and MED30 were shown to have special relevance during the formation of unspliced viral transcripts (p < 0.0005). Knockdown of the selected MED factors compromised HIV transcription induced by Tat, with the strongest inhibitory effect shown by siMED6 and siMED14 cells. Co-immunoprecipitation experiments suggested physical interaction between MED14 and HIV-1 Tat protein. A better understanding of the mechanisms and factors controlling HIV-1 transcription is key to addressing the development of new strategies required to inhibit HIV replication or reactivate HIV-1 from the latent reservoirs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

High resolution top-down experimental strategies on the Orbitrap platform.

PubMed

Scheffler, Kai; Viner, Rosa; Damoc, Eugen

2018-03-20

Top-down mass spectrometry (MS) strategies allow in-depth characterization of proteins by fragmentation of the entire molecule(s) inside a mass spectrometer without requiring prior proteolytic digestion. Importantly, the fragmentation techniques on commercially available mass spectrometers have become more versatile over the past decade, with different characteristics in regards to the type and wealth of fragment ions that can be obtained while preserving labile protein post-translational modifications. Due to these and other improvements, top-down MS has become of broader interest and has started to be applied in more disciplines, such as the quality control of recombinant proteins, analysis and characterization of biopharmaceuticals, and clinical biochemistry to probe protein forms as potential disease biomarkers. This article provides a technical overview and guidance for data acquisition strategies on the Orbitrap platform for single proteins and low complexity protein mixtures. A protein standard mixture composed of six recombinant proteins is also introduced and analysis strategies are discussed in detail. The article provides a detailed overview and guidance on how to choose from the variety of available methods for protein characterization by top-down analysis on the Orbitrap platform. Technical details are provided explaining important observations and phenomena when working with intact proteins and data from a number of different samples should serve to provide a solid understanding on how experiments were and should be setup and to set the right expectations on the outcome of these types of experiments. Additionally, a new intact protein standard sample is introduced that will help as a QC sample to check the instrument's hardware and method setup conditions as a requirement for obtaining high quality data from biologically relevant samples. Copyright © 2017 Elsevier B.V. All rights reserved.

The Reg1-interacting proteins, Bmh1, Bmh2, Ssb1, and Ssb2, have roles in maintaining glucose repression in Saccharomyces cerevisiae.

PubMed

Dombek, Kenneth M; Kacherovsky, Nataly; Young, Elton T

2004-09-10

In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in Deltabmh1Deltabmh2 cells uses the same activation pathway that operates in Deltareg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA(3)-TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA(3)-TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression.

Glycobiology simplified: diverse roles of glycan recognition in inflammation

PubMed Central

Schnaar, Ronald L.

2016-01-01

Glycans and complementary glycan-binding proteins are essential components in the language of cell-cell interactions in immunity. The study of glycan function is the purview of glycobiology, which has often been presented as an unusually complex discipline. In fact, the human glycome, composed of all of its glycans, is built primarily from only 9 building blocks that are combined by enzymes (writers) with specific and limited biosynthetic capabilities into a tractable and increasingly accessible number of potential glycan patterns that are functionally read by several dozen human glycan-binding proteins (readers). Nowhere is the importance of glycan recognition better understood than in infection and immunity, and knowledge in this area has already led to glycan mimetic anti-infective and anti-inflammatory drugs. This review includes a brief tutorial on human glycobiology and a limited number of specific examples of glycan-binding protein-glycan interactions that initiate and regulate inflammation. Examples include representatives from different glycan-binding protein families, including the C-type lectins (E-selectin, P-selectin, dectin-1, and dectin-2), sialic acid-binding immunoglobulin-like lectins (sialic acid-binding immunoglobulin-like lectins 8 and 9), galectins (galectin-1, galectin-3, and galectin-9), as well as hyaluronic acid-binding proteins. As glycoscience technologies advance, opportunities for enhanced understanding of glycans and their roles in leukocyte cell biology provide increasing opportunities for discovery and therapeutic intervention. PMID:27004978

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

PubMed

Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

2009-12-01

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

Insights into the Structure and Function of Ciliary and Flagellar Doublet Microtubules

PubMed Central

Linck, Richard; Fu, Xiaofeng; Lin, Jianfeng; Ouch, Christna; Schefter, Alexandra; Steffen, Walter; Warren, Peter; Nicastro, Daniela

2014-01-01

Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems. PMID:24794867