WorldWideScience

Sample records for ardb proteins reveals

  1. Optical tweezers reveal how proteins alter replication

    Science.gov (United States)

    Chaurasiya, Kathy

    Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

  2. Multifunctional proteins revealed by overlapping clustering in protein interaction network

    OpenAIRE

    Becker, Emmanuelle; Robisson, Benoît; Chapple, Charles E.; Guénoche, Alain; Brun, Christine

    2011-01-01

    Motivation: Multifunctional proteins perform several functions. They are expected to interact specifically with distinct sets of partners, simultaneously or not, depending on the function performed. Current graph clustering methods usually allow a protein to belong to only one cluster, therefore impeding a realistic assignment of multifunctional proteins to clusters. Results: Here, we present Overlapping Cluster Generator (OCG), a novel clustering method which decomposes a network into overla...

  3. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

    Science.gov (United States)

    Shankaran, Mahalakshmi; King, Chelsea L; Angel, Thomas E; Holmes, William E; Li, Kelvin W; Colangelo, Marc; Price, John C; Turner, Scott M; Bell, Christopher; Hamilton, Karyn L; Miller, Benjamin F; Hellerstein, Marc K

    2016-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  4. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins*

    Science.gov (United States)

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina; Cruz-Zaragoza, Luis Daniel; Garcia, Esther; Clausen, Mathias P.; Schliebs, Wolfgang; Erdmann, Ralf; Eggeling, Christian

    2016-01-01

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future. PMID:27311714

  5. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Yingang, E-mail: fengyg@qibebt.ac.cn [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Song, Xiaxia [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Lin, Jinzhong [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xuan, Jinsong [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Cui, Qiu [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Wang, Jinfeng [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  6. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    International Nuclear Information System (INIS)

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae

  7. Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins

    Science.gov (United States)

    Fromont-Racine, Micheline; Mayes, Andrew E.; Brunet-Simon, Adeline; Rain, Jean-Christophe; Colley, Alan; Dix, Ian; Decourty, Laurence; Joly, Nicolas; Ricard, Florence; Beggs, Jean D.

    2000-01-01

    A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale. PMID:10900456

  8. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters. PMID:26371748

  9. Protein painting reveals solvent-excluded drug targets hidden within native protein–protein interfaces

    Science.gov (United States)

    Luchini, Alessandra; Espina, Virginia; Liotta, Lance A.

    2014-01-01

    Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein–protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets. PMID:25048602

  10. Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions.

    Science.gov (United States)

    Shashikanth, Nitesh; Kisting, Meridith A; Leckband, Deborah E

    2016-01-01

    The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions. PMID:27009566

  11. Revealing Higher Order Protein Structure Using Mass Spectrometry

    Science.gov (United States)

    Chait, Brian T.; Cadene, Martine; Olinares, Paul Dominic; Rout, Michael P.; Shi, Yi

    2016-04-01

    The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope.

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

    Science.gov (United States)

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

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. PMID:26659058

  13. Revealing Higher Order Protein Structure Using Mass Spectrometry

    Science.gov (United States)

    Chait, Brian T.; Cadene, Martine; Olinares, Paul Dominic; Rout, Michael P.; Shi, Yi

    2016-06-01

    The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope.

  14. Intragenic suppressor of Osiaa23 revealed a conserved tryptophan residue crucial for protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Jun Ni

    Full Text Available The Auxin/Indole-3-Acetic Acid (Aux/IAA and Auxin Response Factor (ARF are two important families that play key roles in auxin signal transduction. Both of the families contain a similar carboxyl-terminal domain (Domain III/IV that facilitates interactions between these two families. In spite of the importance of protein-protein interactions among these transcription factors, the mechanisms involved in these interactions are largely unknown. In this study, we isolated six intragenic suppressors of an auxin insensitive mutant, Osiaa23. Among these suppressors, Osiaa23-R5 successfully rescued all the defects of the mutant. Sequence analysis revealed that an amino acid substitution occurred in the Tryptophan (W residue in Domain IV of Osiaa23. Yeast two-hybrid experiments showed that the mutation in Domain IV prevents the protein-protein interactions between Osiaa23 and OsARFs. Phylogenetic analysis revealed that the W residue is conserved in both OsIAAs and OsARFs. Next, we performed site-specific amino acid substitutions within Domain IV of OsARFs, and the conserved W in Domain IV was exchanged by Serine (S. The mutated OsARF(WSs can be released from the inhibition of Osiaa23 and maintain the transcriptional activities. Expression of OsARF(WSs in Osiaa23 mutant rescued different defects of the mutant. Our results suggest a previously unknown importance of Domain IV in both families and provide an indirect way to investigate functions of OsARFs.

  15. Two-photon polarization microscopy reveals protein structure and function

    Czech Academy of Sciences Publication Activity Database

    Lazar, Josef; Bondar, Alexey; Timr, S.; Firestein, S. J.

    2011-01-01

    Roč. 8, č. 8 (2011), s. 684-U120. ISSN 1548-7091 Institutional research plan: CEZ:AV0Z60870520 Keywords : green fluorescent protein s * living cells * in-vivo * indicators * anisotropy * activacion * dissociation * orientation * calmodulin * membranes Subject RIV: CE - Biochemistry Impact factor: 19.276, year: 2011

  16. Proteomic and genomic analysis reveals novel Campylobacter jejuni outer membrane proteins and potential heterogeneity

    Directory of Open Access Journals (Sweden)

    Eleanor Watson

    2014-09-01

    Full Text Available Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC–ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith–Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.

  17. Proteomic approach to reveal the proteins associated with encystment of the ciliate Euplotes encysticus.

    Directory of Open Access Journals (Sweden)

    Jiwu Chen

    Full Text Available In order to identify and reveal the proteins related to encystment of the ciliate Euplotes encysticus, we analyzed variation in the abundance of the proteins isolated from the resting cyst comparing with proteins in the vegetative cell. 2-D electrophoresis, MALDI-TOF MS techniques and Bioinformatics were used for proteome separation, quantification and identification. The comparative proteomics studies revealed 26 proteins with changes on the expression in the resting cysts, including 12 specific proteins and 14 differential proteins. 12 specific proteins and 10 out of the 14 differential proteins were selected and identified by MALDI-TOF MS. The identified specific proteins with known functions included type II cytoskeletal 1, keratin, Nop16 domain containing protein, protein arginine n-methyltransferase, epsilon-trimethyllysine hydroxylase and calpain-like protein. The identified differential proteins with known functions included Lysozyme C, keratinocyte growth factor, lysozyme homolog AT-2, formate acetyltransferase, alpha S1 casein and cold-shock protein. We discussed the functions of these proteins as well as their contribution in the process of encystment. These identified proteins covered a wide range of molecular functions, including gene regulation, RNA regulation, proteins degradation and oxidation resistance, stress response, material transport and cytoskeleton organization. Therefore, differential expression of these proteins was essential for cell morphological and physiological changes during encystment. This suggested that the peculiar proteins and differential proteins might play important roles in the process of the vegetative cells transforming into the resting cysts. These observations may be novel findings that bring new insights into the detailed mechanisms of dormancy.

  18. Yeast two hybrid analyses reveal novel binary interactions between human cytomegalovirus-encoded virion proteins.

    Directory of Open Access Journals (Sweden)

    Aaron To

    Full Text Available Human cytomegalovirus (HCMV is the largest human herpesvirus and its virion contains many viral encoded proteins found in the capsid, tegument, and envelope. In this study, we carried out a yeast two-hybrid (YTH analysis to study potential binary interactions among 56 HCMV-encoded virion proteins. We have tested more than 3,500 pairwise combinations for binary interactions in the YTH analysis, and identified 79 potential interactions that involve 37 proteins. Forty five of the 79 interactions were also identified in human cells expressing the viral proteins by co-immunoprecipitation (co-IP experiments. To our knowledge, 58 of the 79 interactions revealed by YTH analysis, including those 24 that were also identified in co-IP experiments, have not been reported before. Novel potential interactions were found between viral capsid proteins and tegument proteins, between tegument proteins, between tegument proteins and envelope proteins, and between envelope proteins. Furthermore, both the YTH and co-IP experiments have identified 9, 7, and 5 interactions that were involved with UL25, UL24, and UL89, respectively, suggesting that these "hub" proteins may function as the organizing centers for connecting multiple virion proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly. Our study provides a framework to study potential interactions between HCMV proteins and investigate the roles of protein-protein interactions in HCMV virion formation or maturation process.

  19. Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins

    OpenAIRE

    Fromont-Racine, Micheline; Mayes, Andrew E.; Brunet-Simon, Adeline; Rain, Jean-Christophe; Colley, Alan; Dix, Ian; Decourty, Laurence; Joly, Nicolas; Ricard, Florence; Beggs, Jean D.; Legrain, Pierre

    2000-01-01

    A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative geno...

  20. Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins

    OpenAIRE

    Fromont-Racine, Micheline; Mayes, Andrew E.; Brunet-Simon, Adeline; Rain, Jean-Christophe; Colley, Alan; Dix, Ian; Decourty, Laurence; Joly, Nicolas; Ricard, Florence; Beggs, Jean D.; Legrain, Pierre

    2000-01-01

    A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (L¯ike Sm¯) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative ge...

  1. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    OpenAIRE

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M.

    2012-01-01

    Rubin and colleagues describe the first structures of full-length and phosphorylated Retinoblastoma (Rb) protein. These structures reveal the mechanism of Rb inactivation and provide valuable insight into this critical tumor suppressor protein's allosteric inhibition via multisite Cdk phosphorylation and its E2F and cell cycle regulation.

  2. Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily.

    Science.gov (United States)

    Yeung, Ho; Squire, Christopher J; Yosaatmadja, Yuliana; Panjikar, Santosh; López, Gemma; Molina, Antonio; Baker, Edward N; Harris, Paul W R; Brimble, Margaret A

    2016-07-01

    Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63-residue member of this family, snakin-1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin-1, determined by a novel combination of racemic protein crystallization and radiation-damage-induced phasing (RIP), is reported. Racemic crystals of snakin-1 and quasi-racemic crystals incorporating an unnatural 4-iodophenylalanine residue were prepared from chemically synthesized d- and l-proteins. Breakage of the C-I bonds in the quasi-racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces. PMID:27145301

  3. Protein Profiling Reveals Novel Proteins in Pollen and Pistil of W22 (ga1; Ga1 in Maize

    Directory of Open Access Journals (Sweden)

    Jin Yu

    2014-05-01

    Full Text Available Gametophytic factors mediate pollen-pistil interactions in maize (Zea mays L. and play active roles in limiting gene flow among maize populations and between maize and teosinte. This study was carried out to identify proteins and investigate the mechanism of gametophytic factors using protein analysis. W22 (ga1; which did not carry a gametophytic factor and W22 (Ga1, a near iso-genic line, were used for the proteome investigation. SDS-PAGE was executed to investigate proteins in the pollen and pistil of W22 (ga1 and W22 (Ga1. A total of 44 differentially expressed proteins were identified in the pollen and pistil on SDS-PAGE using LTQ-FTICR MS. Among the 44 proteins, a total of 24 proteins were identified in the pollen of W22 (ga1 and W22 (Ga1 whereas 20 differentially expressed proteins were identified from the pistil of W22 (ga1 and W22 (Ga1. However, in pollen, 2 proteins were identified only in the W22 (ga1 and 12 proteins only in the W22 (Ga1 whereas 10 proteins were confirmed from the both of W22 (ga1 and W22 (Ga1. In contrary, 10 proteins were appeared only in the pistil of W22 (ga1 and 7 proteins from W22 (Ga1 while 3 proteins confirmed in the both of W22 (ga1 and W22 (Ga1. Moreover, the identified proteins were generally involved in hydrolase activity, nucleic acid binding and nucleotide binding. These results help to reveal the mechanism of gametophytic factors and provide a valuable clue for the pollen and pistil research in maize.

  4. Analysis of protein transport in the Brassica oleracea vasculature reveals protein-specific destinations

    OpenAIRE

    Niu, Chenxing; Anstead, James; Verchot, Jeanmarie

    2012-01-01

    We investigated the vascular transport properties of exogenously applied proteins to Brassica oleracea plants and compared their delivery to various aerial parts of the plant with carboxy fluorescein (CF) dye. We identified unique properties for each protein. Alexafluor-tagged bovine serum albumin (Alexa-BSA) and Alexafluor-tagged Histone H1 (Alexa-Histone) moved slower than CF dye throughout the plant. Interestingly, Alexa-Histone was retained in the phloem and phloem parenchyma while Alexa-...

  5. Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis

    Directory of Open Access Journals (Sweden)

    Kalyan S. Chakrabarti

    2016-01-01

    Full Text Available Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection or are caused in response to ligand binding (induced fit. Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here, we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using nuclear magnetic resonance (NMR spectroscopy, stopped-flow kinetics, and isothermal titration calorimetry, we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding.

  6. Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis.

    Science.gov (United States)

    Chakrabarti, Kalyan S; Agafonov, Roman V; Pontiggia, Francesco; Otten, Renee; Higgins, Matthew K; Schertler, Gebhard F X; Oprian, Daniel D; Kern, Dorothee

    2016-01-01

    Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here, we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using nuclear magnetic resonance (NMR) spectroscopy, stopped-flow kinetics, and isothermal titration calorimetry, we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding. PMID:26725117

  7. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

    Science.gov (United States)

    Kohansal-Nodehi, Mahdokht; Chua, John Je; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. PMID:27115346

  8. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

    Science.gov (United States)

    Dong, Yongbin; Wang, Qilei; Zhang, Long; Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

    2015-01-01

    The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize. PMID:26587848

  9. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

    Directory of Open Access Journals (Sweden)

    Yongbin Dong

    Full Text Available The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

  10. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

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    Robert Kalescky

    2016-04-01

    Full Text Available Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2 in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.

  11. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

    Science.gov (United States)

    Kalescky, Robert; Zhou, Hongyu; Liu, Jin; Tao, Peng

    2016-04-01

    Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery. PMID:27115535

  12. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated.

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    Daehwan Chung

    Full Text Available Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its

  13. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions.

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    Jessica B Hostetler

    2015-12-01

    Full Text Available A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC, and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion.We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins

  14. Protein-protein interaction studies revealed genes associated with plant disease resistance and drought tolerance (abstract)

    International Nuclear Information System (INIS)

    Under natural conditions, plants are frequently subjected to biotic and abiotic constraints that cause considerable damage and limit plant productivity worldwide. Biotic and abiotic stresses results in the accumulation of Reactive Oxygen Species, ROS (H/sub 2/O/sub 2/, O/sub 2/), Nitric oxide (NO) and cytosolic calcium (Ca/sup 2), indicating that plant responses to diseases and drought may operate, at least in part, through common molecular pathways. Additionally, stress-inducible genes have been categorized in two different groups: (a) genes that directly protect against environmental stresses and (b) genes that encode protein kinases intriguingly, protein kinases are also involved in disease resistance since many resistance genes (R genes) are in fact kinases. Here, we describe an interactor hunt using the bacterial virulent gene, VirPphA as a bait to screen an Arabidopsis thaliana cDNA prey library. VirPpha shares sequence similarity with another type III effector protein. AvrPtoB. The screen, originally designed to search for key signaling components involved in disease resistance, identified several putative and promising interactors (2-cys peroxiredoxin-like protein, kinase-like protein and ER6 protein, which is a universal stress protein) that might be involved in both biotic and abiotic stress responses. Simultaneously, another screen using AvrPtoB as a bait was conducted searching the same library for common interactors. Fibrillin (Fibri, At4g04020) was identified in both screens indicating a possible involvement in plant disease resistance through its influence on the plant cytoskeleton, which has been implicated in localized defence response. Furthermore, At4g04020 is 82% similar to the Rice fibrillin, At4g22240, which was recently shown to interact the, rice SGT1 (OsSGT1). SGT1 is a gene that is required for multiple R-gene function. Using the yeast two-hybrid system, fibrillin was found to interact strongly with all VirPphA homologues identified in

  15. Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

    Science.gov (United States)

    Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

    2005-07-01

    Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

  16. The protein architecture of human secretory vesicles reveals differential regulation of signaling molecule secretion by protein kinases.

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    Steven J Bark

    Full Text Available Secretory vesicles are required for release of chemical messengers to mediate intercellular signaling among human biological systems. It is necessary to define the organization of the protein architecture of the 'human' dense core secretory vesicles (DCSV to understand mechanisms for secretion of signaling molecules essential for cellular regulatory processes. This study, therefore, conducted extensive quantitative proteomics and systems biology analyses of human DCSV purified from human pheochromocytoma. Over 600 human DCSV proteins were identified with quantitative evaluation of over 300 proteins, revealing that most proteins participate in producing peptide hormones and neurotransmitters, enzymes, and the secretory machinery. Systems biology analyses provided a model of interacting DCSV proteins, generating hypotheses for differential intracellular protein kinases A and C signaling pathways. Activation of cellular PKA and PKC pathways resulted in differential secretion of neuropeptides, catecholamines, and β-amyloid of Alzheimer's disease for mediating cell-cell communication. This is the first study to define a model of the protein architecture of human DCSV for human disease and health.

  17. Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans

    Science.gov (United States)

    Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C. G.; Benavente, Ricardo

    2012-01-01

    The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed no sequence homology. This discrepancy challenged the hypothesis that the SC arose only once in evolution. To pursue this matter we focused on the evolution of SYCP1 and SYCP3, the two major structural SC proteins of mammals. Remarkably, our comparative bioinformatic and expression studies revealed that SYCP1 and SYCP3 are also components of the SC in the basal metazoan Hydra. In contrast to previous assumptions, we therefore conclude that SYCP1 and SYCP3 form monophyletic groups of orthologous proteins across metazoans. PMID:23012415

  18. Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors

    Science.gov (United States)

    Midde, Krishna K.; Aznar, Nicolas; Laederich, Melanie B.; Ma, Gary S.; Kunkel, Maya T.; Newton, Alexandra C.; Ghosh, Pradipta

    2015-01-01

    Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK–GIV–Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states. PMID:25713130

  19. The integrated analysis of metabolic and protein interaction networks reveals novel molecular organizing principles

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    Walther Dirk

    2008-11-01

    Full Text Available Abstract Background The study of biological interaction networks is a central theme of systems biology. Here, we investigate the relationships between two distinct types of interaction networks: the metabolic pathway map and the protein-protein interaction network (PIN. It has long been established that successive enzymatic steps are often catalyzed by physically interacting proteins forming permanent or transient multi-enzymes complexes. Inspecting high-throughput PIN data, it was shown recently that, indeed, enzymes involved in successive reactions are generally more likely to interact than other protein pairs. In our study, we expanded this line of research to include comparisons of the underlying respective network topologies as well as to investigate whether the spatial organization of enzyme interactions correlates with metabolic efficiency. Results Analyzing yeast data, we detected long-range correlations between shortest paths between proteins in both network types suggesting a mutual correspondence of both network architectures. We discovered that the organizing principles of physical interactions between metabolic enzymes differ from the general PIN of all proteins. While physical interactions between proteins are generally dissortative, enzyme interactions were observed to be assortative. Thus, enzymes frequently interact with other enzymes of similar rather than different degree. Enzymes carrying high flux loads are more likely to physically interact than enzymes with lower metabolic throughput. In particular, enzymes associated with catabolic pathways as well as enzymes involved in the biosynthesis of complex molecules were found to exhibit high degrees of physical clustering. Single proteins were identified that connect major components of the cellular metabolism and may thus be essential for the structural integrity of several biosynthetic systems. Conclusion Our results reveal topological equivalences between the protein

  20. Hydrogen bonds of DsrD protein revealed by neutron crystallography

    International Nuclear Information System (INIS)

    Hydrogen bonds of DNA-binding protein DsrD have been determined by neutron diffraction. In terms of proton donors and acceptors, DsrD protein shows striking differences from other proteins. The features of hydrogen bonds in DsrD protein from sulfate-reducing bacteria have been investigated by neutron protein crystallography. The function of DsrD has not yet been elucidated clearly, but its X-ray crystal structure revealed that it comprises a winged-helix motif and shows the highest structural homology to the DNA-binding proteins. Since any neutron structure of a DNA recognition protein has not yet been obtained, here detailed information on the hydrogen bonds in the winged-helix-motif protein is given and the following features found. (i) The number of hydrogen bonds per amino acid of DsrD is relatively fewer than for other proteins for which neutron structures were determined previously. (ii) Hydrogen bonds are localized between main-chain and main-chain atoms; there are few hydrogen bonds between main-chain and side-chain atoms and between side-chain and side-chain atoms. (iii) Hydrogen bonds inducted by protonation of specific amino acid residues (Glu50) seem to play an essential role in the dimerization of DsrD. The former two points are related to the function of the DNA-binding protein; the three-dimensional structure was mainly constructed by hydrogen bonds in main chains, while the side chains appeared to be used for another role. The latter point would be expected to contribute to the crystal growth of DsrD

  1. Proteomic analysis of a segregant population reveals candidate proteins linked to mealiness in peach.

    Science.gov (United States)

    Almeida, Andréa Miyasaka; Urra, Claudio; Moraga, Carol; Jego, Marcela; Flores, Alejandra; Meisel, Lee; González, Mauricio; Infante, Rodrigo; Defilippi, Bruno G; Campos-Vargas, Reinaldo; Orellana, Ariel

    2016-01-10

    Peaches are stored at low temperatures to delay ripening and increase postharvest life. However some varieties are susceptible to chilling injury,which leads to fruit mealiness, browning and flesh bleeding. In order to identify potentialmarkers associated with chilling injury,we performed proteomic analyses on a segregating population with contrasting susceptibility to chilling-induced mealiness. Chilling-induced mealiness was assessed by measuring juiciness in fruits that have been stored in cold and then allowed to ripen. Fruitmesocarp and leaf proteome from contrasting segregants were analyzed using 2-DE gels. Comparison of protein abundance between segregants revealed 133 spots from fruit mesocarp and 36 from leaf. Thirty four fruit mesocarp proteins were identified from these spots. Most of these proteins were related to ethylene synthesis, ABA response and stress response. Leaf protein analyses identified 22 proteins, most of which related to energy metabolism. Some of the genes that code for these proteins have been previously correlated with chilling injury through transcript analyses and co-segregation with mealiness QTLs. The results from this study, further deciphers the molecular mechanisms associated with chilling response in peach fruit, and identifies candidate proteins linked to mealiness in peach which may be used as putative markers for this trait. PMID:26459401

  2. Systematic protein interactome analysis of glycosaminoglycans revealed YcbS as a novel bacterial virulence factor.

    Science.gov (United States)

    Hsiao, Felix Shih-Hsiang; Sutandy, Fx Reymond; Syu, Guan-Da; Chen, Yi-Wen; Lin, Jun-Mu; Chen, Chien-Sheng

    2016-01-01

    Microbial pathogens have evolved several strategies for interacting with host cell components, such as glycosaminoglycans (GAGs). Some microbial proteins involved in host-GAG binding have been described; however, a systematic study on microbial proteome-mammalian GAG interactions has not been conducted. Here, we used Escherichia coli proteome chips to probe four typical mammalian GAGs, heparin, heparan sulphate (HS), chondroitin sulphate B (CSB), and chondroitin sulphate C (CSC), and identified 185 heparin-, 62 HS-, 98 CSB-, and 101 CSC-interacting proteins. Bioinformatics analyses revealed the unique functions of heparin- and HS-specific interacting proteins in glycine, serine, and threonine metabolism. Among all the GAG-interacting proteins, three were outer membrane proteins (MbhA, YcbS, and YmgH). Invasion assays confirmed that mutant E. coli lacking ycbS could not invade the epithelial cells. Introducing plasmid carrying ycbS complemented the invading defects at ycbS lacking E. coli mutant, that can be further improved by overexpressing ycbS. Preblocking epithelial cells with YcbS reduced the percentage of E. coli invasions. Moreover, we observed that whole components of the ycb operon were crucial for invasion. The displacement assay revealed that YcbS binds to the laminin-binding site of heparin and might affect the host extracellular matrix structure by displacing heparin from laminin. PMID:27323865

  3. Coarse grained simulation reveals antifreeze properties of hyperactive antifreeze protein from Antarctic bacterium Colwellia sp.

    Science.gov (United States)

    Nguyen, Hung; Van, Thanh Dac; Le, Ly

    2015-10-01

    The novel hyperactive antifreeze protein (AFP) of Antarctic sea ice bacterium Colwellia sp. provides a target for studying the protection of psychrophilic microgoranisms against freezing environment. Interestingly, the Colwellia sp. hyperactive antifreeze protein (ColAFP) was crystallized without the structural dynamic characteristics. Here, the result indicated, through coarse grained simulation of ColAFP under various subfreezing temperature, that ColAFP remains active at temperature of equal and greater than 275 K (∼2 °C). Extensive simulation analyses also revealed the adaptive mechanism of ColAFP in subfreezing environment. Our result provides a structural dynamic understanding of the ColAFP.

  4. Dynamic Coupling among Protein Binding, Sliding, and DNA Bending Revealed by Molecular Dynamics.

    Science.gov (United States)

    Tan, Cheng; Terakawa, Tsuyoshi; Takada, Shoji

    2016-07-13

    Protein binding to DNA changes the DNA's structure, and altered DNA structure can, in turn, modulate the dynamics of protein binding. This mutual dependency is poorly understood. Here we investigated dynamic couplings among protein binding to DNA, protein sliding on DNA, and DNA bending by applying a coarse-grained simulation method to the bacterial architectural protein HU and 14 other DNA-binding proteins. First, we verified our method by showing that the simulated HU exhibits a weak preference for A/T-rich regions of DNA and a much higher affinity for gapped and nicked DNA, consistent with biochemical experiments. The high affinity was attributed to a local DNA bend, but not the specific chemical moiety of the gap/nick. The long-time dynamic analysis revealed that HU sliding is associated with the movement of the local DNA bending site. Deciphering single sliding steps, we found the coupling between HU sliding and DNA bending is akin to neither induced-fit nor population-shift; instead they moved concomitantly. This is reminiscent of a cation transfer on DNA and can be viewed as a protein version of polaron-like sliding. Interestingly, on shorter time scales, HU paused when the DNA was highly bent at the bound position and escaped from pauses once the DNA spontaneously returned to a less bent structure. The HU sliding is largely regulated by DNA bending dynamics. With 14 other proteins, we explored the generality and versatility of the dynamic coupling and found that 6 of the 15 assayed proteins exhibit the polaron-like sliding. PMID:27309278

  5. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

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    Huang Yong

    2009-11-01

    Full Text Available Abstract Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64% consisted of both mitochondrial and cytosolic (non-mitochondrial proteins (mt-cy families while the remaining 33 (36% were composed of mitochondrial proteins (mt-mt families. Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1 relocalization from mitochondria to cytosol, 2 from cytosol to mitochondria and 3 multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on

  6. Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

    Science.gov (United States)

    Hamsten, C; Häggmark, A; Grundström, J; Mikus, M; Lindskog, C; Konradsen, J R; Eklund, A; Pershagen, G; Wickman, M; Grunewald, J; Melén, E; Hedlin, G; Nilsson, P; van Hage, M

    2016-09-01

    Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma. PMID:27145233

  7. Proteomic Profiling Reveals Upregulated Protein Expression of Hsp70 in Keloids

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    Ju Hee Lee

    2013-01-01

    Full Text Available Background. The biochemical characteristics of keloid-derived fibroblasts differ from those of adjacent normal fibroblasts, and these differences are thought to be the cause of abnormal fibrosis. Therefore, we investigated the characteristic proteins that are differentially expressed in keloid-derived fibroblasts using proteomics tools. Objective. We attempted to investigate the novel proteins that play important roles in the pathophysiology of keloids. Methods. Proteomics analysis was performed to identify differentially expressed proteins in keloid-derived fibroblasts. Keloid-derived fibroblasts and adjacent normal fibroblasts were analyzed with 2-DAGE. We validated these proteins with immunoblot analysis, real-time RT-PCR, and immunohistochemistry. Results. Sixteen differentially expressed protein spots were identified in keloid-derived fibroblasts. Among them, heat shock protein 70 (Hsp70 was specifically upregulated in keloid-derived fibroblasts. Also, immunohistochemistry and western blot analysis revealed increased Hsp70, TGF-β, and PCNA expressions in keloids compared to normal tissue. Conclusion. Hsp70 is overexpressed in keloid fibroblasts and tissue. The overexpression of Hsp70 may be involved in the pathogenesis of keloids, and the inhibition of Hsp70 could be a new therapeutic tool for the treatment of keloids.

  8. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

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    Nordlund Henri R

    2005-03-01

    Full Text Available Abstract Background A chicken egg contains several biotin-binding proteins (BBPs, whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

  9. Proteome analysis of Paenibacillus larvae reveals the existence of a putative S-layer protein.

    Science.gov (United States)

    Fünfhaus, Anne; Genersch, Elke

    2012-04-01

    Honey bee pathology has attracted much interest recently due to the problems with honey bee declines in many regions of the world. American Foulbrood (AFB) caused by Paenibacillus larvae is the most devastating bacterial brood disease of the Western honey bee (Apis mellifera) causing considerable economic losses to beekeepers worldwide. AFB outbreaks are mainly caused by two differentially virulent genotypes of P. larvae, P. larvae ERIC I and ERIC II. To better understand AFB pathogenesis and to complement already existing data from the genetic level we aimed at obtaining expression data from the protein level. We successfully developed a protocol for two-dimensional proteome analysis of P. larvae with subsequent mass-spectrometry based protein sequencing. Based on the obtained master protein maps of P. larvae genotypes ERIC I and II we identified the dominantly expressed cytosolic proteins of both genotypes, some of them presumably linked to pathogenesis and virulence. Comparing the master maps of both genotypes revealed differentially expressed proteins, i.e. a putative S-layer protein which is expressed by P. larvae ERIC II but absent from the proteome of P. larvae ERIC I. The implications of our findings for pathogenesis of AFB and virulence of P. larvae will be discussed. PMID:23757273

  10. Proteomic analysis reveals differentially regulated protein acetylation in human amyotrophic lateral sclerosis spinal cord.

    Directory of Open Access Journals (Sweden)

    Dong Liu

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive fatal neurodegenerative disease that primarily affects motor neurons in the brain and spinal cord. Histone deacetylase (HDAC inhibitors have neuroprotective effects potentially useful for the treatment of neurodegenerative diseases including ALS; however, the molecular mechanisms underlying their potential efficacy is not well understood. Here we report that protein acetylation in urea-soluble proteins is differently regulated in post-mortem ALS spinal cord. Two-dimensional electrophoresis (2-DE analysis reveals several protein clusters with similar molecular weight but different charge status. Liquid chromatography and tandem mass spectrometry (LC-MS/MS identifies glial fibrillary acidic protein (GFAP as the dominant component in the protein clusters. Further analysis indicates six heavily acetylated lysine residues at positions 89, 153, 189, 218, 259 and 331 of GFAP. Immunoprecipitation followed by Western blotting confirms that the larger form of GFAP fragments are acetylated and upregulated in ALS spinal cord. Further studies demonstrate that acetylation of the proteins additional to GFAP is differently regulated, suggesting that acetylation and/or deacetylation play an important role in pathogenesis of ALS.

  11. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

    Science.gov (United States)

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D

    2016-03-22

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  13. The Plasmodium berghei translocon of exported proteins reveals spatiotemporal dynamics of tubular extensions.

    Science.gov (United States)

    Matz, Joachim M; Goosmann, Christian; Brinkmann, Volker; Grützke, Josephine; Ingmundson, Alyssa; Matuschewski, Kai; Kooij, Taco W A

    2015-01-01

    The erythrocyte is an extraordinary host cell for intracellular pathogens and requires extensive remodelling to become permissive for infection. Malaria parasites modify their host red blood cells through protein export to acquire nutrients and evade immune responses. Endogenous fluorescent tagging of three signature proteins of the Plasmodium berghei translocon of exported proteins (PTEX), heat shock protein 101, exported protein 2 (EXP2), and PTEX88, revealed motile, tubular extensions of the parasitophorous vacuole that protrude from the parasite far into the red blood cell. EXP2 displays a more prominent presence at the periphery of the parasite, consistent with its proposed role in pore formation. The tubular compartment is most prominent during trophozoite growth. Distinct spatiotemporal expression of individual PTEX components during sporogony and liver-stage development indicates additional functions and tight regulation of the PTEX translocon during parasite life cycle progression. Together, live cell imaging and correlative light and electron microscopy permitted previously unrecognized spatiotemporal and subcellular resolution of PTEX-containing tubules in murine malaria parasites. These findings further refine current models for Plasmodium-induced erythrocyte makeover. PMID:26219962

  14. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

    Science.gov (United States)

    Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

    2016-06-01

    Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria. PMID:27183143

  15. Effects of pressure on the dynamics of a hyperthermophilic protein revealed by quasielastic neutron scattering

    Science.gov (United States)

    Shrestha, U. R.; Bhowmik, D.; Copley, J. R. D.; Tyagi, M.; Leao, J. B.; Chu, X.-Q.

    Inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens is a large oligomeric protein derived from hyperthermophilic microorganism that is found near hydrothermal vents deep under the sea, where the pressure is nearly 100 MPa. Here we study the effects of pressure on the conformational flexibility and relaxation dynamics of IPPase over a wide temperature range using quasielastic neutron scattering (QENS) technique. Two spectrometers were used to investigate the β-relaxation dynamics of proteins in time ranges from 2 to 25 ps, and from 100 ps to 2 ns. Our results reveal that, under the pressure of 100 MPa, IPPase displays much faster relaxation dynamics than a mesophilic model protein, hen egg white lysozyme (HEWL), opposite to what we observed previously under the ambient pressure. These contradictory observations imply that high pressure affects the dynamical properties of proteins by distorting their energy landscapes. Accordingly, we derived a general schematic denaturation phase diagram that can be used as a general picture to understand the effects of pressure on protein dynamics and activities Wayne State Univ Startup Fund.

  16. Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus.

    Science.gov (United States)

    Huang, Mingwei; Hebert, Alexander S; Coon, Joshua J; Hull, Christina M

    2015-08-01

    Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote. PMID:26313153

  17. Molecular Dynamics Simulation Reveals Correlated Inter-Lobe Motion in Protein Lysine Methyltransferase SMYD2

    Science.gov (United States)

    Spellmon, Nicholas; Sun, Xiaonan; Sirinupong, Nualpun; Edwards, Brian; Li, Chunying; Yang, Zhe

    2015-01-01

    SMYD proteins are an exciting field of study as they are linked to many types of cancer-related pathways. Cardiac and skeletal muscle development and function also depend on SMYD proteins opening a possible avenue for cardiac-related treatment. Previous crystal structure studies have revealed that this special class of protein lysine methyltransferases have a bilobal structure, and an open–closed motion may regulate substrate specificity. Here we use the molecular dynamics simulation to investigate the still-poorly-understood SMYD2 dynamics. Cross-correlation analysis reveals that SMYD2 exhibits a negative correlated inter-lobe motion. Principle component analysis suggests that this correlated dynamic is contributed to by a twisting motion of the C-lobe with respect to the N-lobe and a clamshell-like motion between the lobes. Dynamical network analysis defines possible allosteric paths for the correlated dynamics. There are nine communities in the dynamical network with six in the N-lobe and three in the C-lobe, and the communication between the lobes is mediated by a lobe-bridging β hairpin. This study provides insight into the dynamical nature of SMYD2 and could facilitate better understanding of SMYD2 substrate specificity. PMID:26717235

  18. The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function

    International Nuclear Information System (INIS)

    The crystal structure of the BVU2987 gene product from B. vulgatus (UniProt A6L4L1) reveals that members of the new Pfam family PF11396 (domain of unknown function; DUF2874) are similar to β-lactamase inhibitor protein and YpmB. Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA-OmlA proteins and hence are likely to function as inhibitory proteins

  19. Nullspace Sampling with Holonomic Constraints Reveals Molecular Mechanisms of Protein Gαs.

    Directory of Open Access Journals (Sweden)

    Dimitar V Pachov

    2015-07-01

    Full Text Available Proteins perform their function or interact with partners by exchanging between conformational substates on a wide range of spatiotemporal scales. Structurally characterizing these exchanges is challenging, both experimentally and computationally. Large, diffusional motions are often on timescales that are difficult to access with molecular dynamics simulations, especially for large proteins and their complexes. The low frequency modes of normal mode analysis (NMA report on molecular fluctuations associated with biological activity. However, NMA is limited to a second order expansion about a minimum of the potential energy function, which limits opportunities to observe diffusional motions. By contrast, kino-geometric conformational sampling (KGS permits large perturbations while maintaining the exact geometry of explicit conformational constraints, such as hydrogen bonds. Here, we extend KGS and show that a conformational ensemble of the α subunit Gαs of heterotrimeric stimulatory protein Gs exhibits structural features implicated in its activation pathway. Activation of protein Gs by G protein-coupled receptors (GPCRs is associated with GDP release and large conformational changes of its α-helical domain. Our method reveals a coupled α-helical domain opening motion while, simultaneously, Gαs helix α5 samples an activated conformation. These motions are moderated in the activated state. The motion centers on a dynamic hub near the nucleotide-binding site of Gαs, and radiates to helix α4. We find that comparative NMA-based ensembles underestimate the amplitudes of the motion. Additionally, the ensembles fall short in predicting the accepted direction of the full activation pathway. Taken together, our findings suggest that nullspace sampling with explicit, holonomic constraints yields ensembles that illuminate molecular mechanisms involved in GDP release and protein Gs activation, and further establish conformational coupling between key

  20. Interplay of protein and DNA structure revealed in simulations of the lac operon.

    Directory of Open Access Journals (Sweden)

    Luke Czapla

    Full Text Available The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

  1. Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis

    DEFF Research Database (Denmark)

    Kjærsgård, Inger Vibeke Holst; Nørrelykke, M.R.; Jessen, Flemming

    2006-01-01

    myosin light chain 1, 2 and 3, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase A and two ?-actin fragments, and a nuclease diphosphate kinase B fragment to change in concentration, during frozen storage. Application of proteomics, multivariate data analysis and MS/MS to......Multivariate data analysis has been combined with proteomics to enhance the recovery of information from 2-DE of cod muscle proteins during different storage conditions. Proteins were extracted according to 11 different storage conditions and samples were resolved by 2-DE. Data generated by 2-DE...... was subjected to principal component analysis (PCA) and discriminant partial least squares regression (DPLSR). Applying PCA to 2-DE data revealed the samples to form groups according to frozen storage time, whereas differences due to different storage temperatures or chilled storage in modified...

  2. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

    Science.gov (United States)

    Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

    2015-10-13

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

  3. Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Hast, Michael A.; Nichols, Connie B.; Armstrong, Stephanie M.; Kelly, Shannon M.; Hellinga, Homme W.; Alspaugh, J. Andrew; Beese, Lorena S. (Duke)

    2012-09-17

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

  4. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity

    Science.gov (United States)

    2016-01-01

    ABSTRACT Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  5. QUANTUM DOT SINGLE MOLECULE TRACKING REVEALS A WIDE RANGE OF DIFFUSIVE MOTIONS OF MEMBRANE TRANSPORT PROTEINS.

    Science.gov (United States)

    Crane, Jonathan M; Haggie, Peter M; Verkman, A S

    2009-03-01

    Single particle tracking (SPT) provides information about the microscopic motions of individual particles in live cells. We applied SPT to study the diffusion of membrane transport proteins in cell plasma membranes in which individual proteins are labeled with quantum dots at engineered extracellular epitopes. Software was created to deduce particle diffusive modes from quantum dot trajectories. SPT of aquaporin (AQP) water channels and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels revealed several types of diffusion. AQP1 was freely mobile in cell membranes, showing rapid, Brownian-type diffusion. The full-length (M1) isoform of AQP4 also diffused rapidly, though the diffusion of a shorter (M23) isoform of AQP4 was highly restricted due to its supermolecular assembly in raft-like orthogonal arrays. CFTR mobility was also highly restricted, in a spring-like potential, due to its tethering to the actin cytoskeleton through PDZ-domain C-terminus interactions. The biological significance of regulated diffusion of membrane transport proteins is a subject of active investigation. PMID:24386532

  6. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    Energy Technology Data Exchange (ETDEWEB)

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M. (UCSC); (LBNL)

    2012-07-18

    Cyclin-dependent kinase (Cdk) phosphorylation of the Retinoblastoma protein (Rb) drives cell proliferation through inhibition of Rb complexes with E2F transcription factors and other regulatory proteins. We present the first structures of phosphorylated Rb that reveal the mechanism of its inactivation. S608 phosphorylation orders a flexible 'pocket' domain loop such that it mimics and directly blocks E2F transactivation domain (E2F{sup TD}) binding. T373 phosphorylation induces a global conformational change that associates the pocket and N-terminal domains (RbN). This first multidomain Rb structure demonstrates a novel role for RbN in allosterically inhibiting the E2F{sup TD}-pocket association and protein binding to the pocket 'LxCxE' site. Together, these structures detail the regulatory mechanism for a canonical growth-repressive complex and provide a novel example of how multisite Cdk phosphorylation induces diverse structural changes to influence cell cycle signaling.

  7. Purification of reversibly oxidized proteins (PROP reveals a redox switch controlling p38 MAP kinase activity.

    Directory of Open Access Journals (Sweden)

    Dennis J Templeton

    Full Text Available Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.

  8. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  9. Atomic force microscopy reveals the mechanical design of a modular protein

    OpenAIRE

    Li, Hongbin; Oberhauser, Andres F.; Fowler, Susan B.; Clarke, Jane; Fernandez, Julio M.

    2000-01-01

    Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanic...

  10. Characterization of the autophagy marker protein Atg8 reveals atypical features of autophagy in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Rahul Navale

    Full Text Available Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8 employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64 and aspartic (pepstatin protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine, indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in

  11. Comparative Genome Sequence Analysis Reveals the Extent of Diversity and Conservation for Glycan-Associated Proteins in Burkholderia spp.

    OpenAIRE

    Ong, Hui San; Mohamed, Rahmah; Firdaus-Raih, Mohd

    2012-01-01

    Members of the Burkholderia family occupy diverse ecological niches. In pathogenic family members, glycan-associated proteins are often linked to functions that include virulence, protein conformation maintenance, surface recognition, cell adhesion, and immune system evasion. Comparative analysis of available Burkholderia genomes has revealed a core set of 178 glycan-associated proteins shared by all Burkholderia of which 68 are homologous to known essential genes. The genome sequence compari...

  12. Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma

    Science.gov (United States)

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-11-01

    Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance.

  13. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    Science.gov (United States)

    He, Yi-Ming; Ma, Bin-Guang

    2016-05-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

  14. Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila heterochromatin protein 1 (HP1 gene family.

    Directory of Open Access Journals (Sweden)

    Mia T Levine

    Full Text Available Heterochromatin is the gene-poor, satellite-rich eukaryotic genome compartment that supports many essential cellular processes. The functional diversity of proteins that bind and often epigenetically define heterochromatic DNA sequence reflects the diverse functions supported by this enigmatic genome compartment. Moreover, heterogeneous signatures of selection at chromosomal proteins often mirror the heterogeneity of evolutionary forces that act on heterochromatic DNA. To identify new such surrogates for dissecting heterochromatin function and evolution, we conducted a comprehensive phylogenomic analysis of the Heterochromatin Protein 1 gene family across 40 million years of Drosophila evolution. Our study expands this gene family from 5 genes to at least 26 genes, including several uncharacterized genes in Drosophila melanogaster. The 21 newly defined HP1s introduce unprecedented structural diversity, lineage-restriction, and germline-biased expression patterns into the HP1 family. We find little evidence of positive selection at these HP1 genes in both population genetic and molecular evolution analyses. Instead, we find that dynamic evolution occurs via prolific gene gains and losses. Despite this dynamic gene turnover, the number of HP1 genes is relatively constant across species. We propose that karyotype evolution drives at least some HP1 gene turnover. For example, the loss of the male germline-restricted HP1E in the obscura group coincides with one episode of dramatic karyotypic evolution, including the gain of a neo-Y in this lineage. This expanded compendium of ovary- and testis-restricted HP1 genes revealed by our study, together with correlated gain/loss dynamics and chromosome fission/fusion events, will guide functional analyses of novel roles supported by germline chromatin.

  15. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    OpenAIRE

    Yi-Ming He; Bin-Guang Ma

    2016-01-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophil...

  16. Unintended Changes in Genetically Modified Rice Expressing the Lysine-Rich Fusion Protein Gene Revealed by a Proteomics Approach

    Institute of Scientific and Technical Information of China (English)

    ZHAO Xiang-xiang; TANG Tang; LIU Fu-xia; LU Chang-li; HU Xiao-lan; JI Li-lian; LIU Qiao-quan

    2013-01-01

    Development of new technologies for evaluating genetically modiifed (GM) crops has revealed that there are unintended insertions and expression changes in GM crops. Proifling techniques are non-targeted approaches and are capable of detecting more unintended changes in GM crops. Here, we report the application of a comparative proteomic approach to investigate the protein proifle differences between a GM rice line, which has a lysine-rich protein gene, and its non-transgenic parental line. Proteome analysis by two-dimensional gel electrophoresis (2-DE) and mass spectrum analysis of the seeds identiifed 22 differentially expressed protein spots. Apart from a number of glutelins that were detected as targeted proteins in the GM line, the majority of the other changed proteins were involved in carbohydrate metabolism, protein synthesis and stress responses. These results indicated that the altered proteins were not associated with plant allergens or toxicity.

  17. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function.

    Science.gov (United States)

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  18. Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation

    Energy Technology Data Exchange (ETDEWEB)

    Raymond, Donald D.; Piper, Mary E.; Gerrard, Sonja R.; Smith, Janet L. (Michigan)

    2010-07-13

    Rift Valley fever virus (RVFV) is a negative-sense RNA virus (genus Phlebovirus, family Bunyaviridae) that infects livestock and humans and is endemic to sub-Saharan Africa. Like all negative-sense viruses, the segmented RNA genome of RVFV is encapsidated by a nucleocapsid protein (N). The 1.93-{angstrom} crystal structure of RVFV N and electron micrographs of ribonucleoprotein (RNP) reveal an encapsidated genome of substantially different organization than in other negative-sense RNA virus families. The RNP polymer, viewed in electron micrographs of both virus RNP and RNP reconstituted from purified N with a defined RNA, has an extended structure without helical symmetry. N-RNA species of {approx}100-kDa apparent molecular weight and heterogeneous composition were obtained by exhaustive ribonuclease treatment of virus RNP, by recombinant expression of N, and by reconstitution from purified N and an RNA oligomer. RNA-free N, obtained by denaturation and refolding, has a novel all-helical fold that is compact and well ordered at both the N and C termini. Unlike N of other negative-sense RNA viruses, RVFV N has no positively charged surface cleft for RNA binding and no protruding termini or loops to stabilize a defined N-RNA oligomer or RNP helix. A potential protein interaction site was identified in a conserved hydrophobic pocket. The nonhelical appearance of phlebovirus RNP, the heterogeneous {approx}100-kDa N-RNA multimer, and the N fold differ substantially from the RNP and N of other negative-sense RNA virus families and provide valuable insights into the structure of the encapsidated phlebovirus genome.

  19. Postnatal development of cerebellar zones revealed by neurofilament heavy chain protein expression

    Directory of Open Access Journals (Sweden)

    Joshua J White

    2013-05-01

    Full Text Available The cerebellum is organized into parasagittal zones that control sensory-motor behavior. Although the architecture of adult zones is well understood, very little is known about how zones emerge during development. Understanding the process of zone formation is an essential step towards unraveling how circuits are constructed to support specific behaviors. Therefore, we focused this study on postnatal development to determine the spatial and temporal changes that establish zonal patterns during circuit formation. We used a combination of wholemount and tissue section immunohistochemistry in mice to show that the cytoskeletal protein neurofilament heavy chain (NFH is a robust marker for postnatal cerebellar zonal patterning. The patterned expression of NFH is initiated shortly after birth, and compared to the domains of several known zonal markers such as zebrin II, HSP25, neurogranin, and phospholipase Cβ4 (PLCβ4, NFH does not exhibit transient expression patterns that are typically remodeled between stages, and the adult zones do not emerge after a period of uniform expression in all lobules. Instead, we found that throughout postnatal development NFH gradually reveals distinct zones in each cerebellar lobule. The boundaries of individual NFH zones sharpen over time, as zones are refined during the second and third weeks after birth. Double labeling with neurogranin and PLCβ4 further revealed that although the postnatal expression of NFH is spatially and temporally unique, its pattern of zones respects a fundamental and well-known molecular topography in the cerebellum. The dynamics of NFH expression support the hypothesis that adult circuits are derived from an embryonic map that is refined into zones during the first three-weeks of life.

  20. Super-Resolution Microscopy Reveals Altered Desmosomal Protein Organization in Tissue from Patients with Pemphigus Vulgaris.

    Science.gov (United States)

    Stahley, Sara N; Warren, Maxine F; Feldman, Ron J; Swerlick, Robert A; Mattheyses, Alexa L; Kowalczyk, Andrew P

    2016-01-01

    Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease in which autoantibodies (IgG) are directed against the desmosomal cadherin desmoglein 3. To better understand how PV IgG alters desmosome morphology and function in vivo, biopsies from patients with PV were analyzed by structured illumination microscopy, a form of superresolution fluorescence microscopy. In patient tissue, desmosomal proteins were aberrantly clustered and patient IgG colocalized with markers for lipid rafts and endosomes. Additionally, steady-state levels of desmoglein 3 were decreased and desmosomes were reduced in size in patient tissue. Desmosomes at blister sites were occasionally split, with PV IgG decorating the extracellular faces of split desmosomes. Desmosome splitting was recapitulated in vitro by exposing cultured keratinocytes both to PV IgG and to mechanical stress, demonstrating that splitting at the blister interface in patient tissue is due to compromised desmosomal adhesive function. These findings indicate that desmoglein 3 clustering and endocytosis are associated with reduced desmosome size and adhesion defects in tissue of patients with PV. Further, this study reveals that superresolution optical imaging is a powerful approach for studying epidermal adhesion structures in normal and diseased skin. PMID:26763424

  1. Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli.

    Science.gov (United States)

    Johnson, Brant R; Hymes, Jeffrey; Sanozky-Dawes, Rosemary; Henriksen, Emily DeCrescenzo; Barrangou, Rodolphe; Klaenhammer, Todd R

    2016-01-01

    The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa. PMID:26475115

  2. Differential appearance of isoforms and cultivar variation in protein temporal profiles revealed in the maturing barley grain proteome

    DEFF Research Database (Denmark)

    Finnie, Christine; Bak-Jensen, K.S.; Laugesen, Sabrina; Roepstorff, P.; Svensson, Birte

    2006-01-01

    Proteome analysis of mature barley (Hordeum vulgare subsp. vulgare) seeds has led to the identification of proteins in about 450 spots on 2D-gels. To shed light on the role of some of these proteins, their temporal appearance was monitored over 5 weeks during grain-filling and maturation of field......-grown barley. Appearance profiles are described for 105 proteins identified in 185 2D-gel spots in the overlapping pI ranges 4-7 and 6-11. Grouping of proteins according to appearance across functional categories revealed instances of differential regulation of protein forms. Thus, a single 1-cys...... modification or defence against fungal pathogens. Comparison of two cultivars, Barke and Morex, led to identification of protein spots appearing earlier in Morex than Barke, reflecting the faster maturation of Morex seeds....

  3. Specific and nonspecific interactions in ultraweak protein−protein associations revealed by solvent paramagnetic relaxation enhancements

    DEFF Research Database (Denmark)

    Johansson, Helle; Jensen, Malene Ringkjøbing; Gesmar, Henrik;

    2014-01-01

    Weak and transient protein–protein interactions underlie numerous biological processes. However, the location of the interaction sites of the specific complexes and the effect of transient, non-specific protein–protein interactions often remain elusive. We have investigated the weak selfassociation...... relaxation agent, it is shown that a distinction can be made between residues that are affected only by transient, non-specific protein–protein interactions and residues that are involved in specific protein-protein associations. Thus, the PREs of the former residues increase linearly with the h......GH concentration in the entire concentration range because of a reduction of the diffusion caused by the transient, non-specific protein-protein interactions, while the PREs of the latter residues increase only at the lower hGH concentrations but decrease at the higher concentrations because of specific protein-protein...

  4. An unbiased nuclear proteomics approach reveals novel nuclear protein components that participates in MAMP-triggered immunity.

    Science.gov (United States)

    Fakih, Zainab; Ahmed, Md Bulbul; Letanneur, Claire; Germain, Hugo

    2016-06-01

    (MAMP)-triggered immunity (MTI) is the first layer of molecular defense encountered by pathogens. Genetic screens have contributed to our knowledge of MTI, but are limited to phenotype-causing mutations. Here we attempt to identify novel factors involved in the early event leading to plant MTI by comparing the nuclear proteomes of two Arabidopsis genotypes treated with chitosan. Our approach revealed that following chitosan treatment, cerk1 plants had many nuclear accumulating proteins in common, but also some unique ones, when compared with Col-0 plants. Analysis of the identified proteins revealed a nuclear accumulation of DNA-modifying enzymes, RNA-binding proteins and ribosomal proteins. Our results demonstrate that nuclear proteomic is a valid, phenotype-independent approach to uncover factor involved in cellular processes. PMID:27177187

  5. Changes in the starch-protein interface depending on common wheat grain hardness revealed using atomic force microscopy

    OpenAIRE

    Chichti, Emna; GEORGE, MATTHIEU; Delenne, Jean-Yves; Lullien-pellerin, Valerie

    2015-01-01

    The Atomic Force Microscope tip was used to progressively abrade the surface of non-cutted starch granules embedded in the endosperm protein matrix in grain sections from wheat near-isogenic lines differing in the puroindoline b gene and thus hardness. In the hard near-isogenic wheat lines, starch granules exhibited two distinct profiles corresponding either to abrasion in the surrounding protein layer or the starch granule. An additional profile, only identified in soft lines, revealed a mar...

  6. Simulations of HIV Capsid Protein Dimerization Reveal the Effect of Chemistry and Topography on the Mechanism of Hydrophobic Protein Association

    Science.gov (United States)

    Yu, Naiyin; Hagan, Michael F.

    2012-09-01

    Recent work has shown that the hydrophobic protein surfaces in aqueous solution sit near a drying transition. The tendency for these surfaces to expel water from their vicinity leads to self assembly of macromolecular complexes. In this article we show with a realistic model for a biologically pertinent system how this phenomenon appears at the molecular level. We focus on the association of the C-terminal domain (CA-C) of the human immunodeficiency virus (HIV) capsid protein. By combining all-atom simulations with specialized sampling techniques we measure the water density distribution during the approach of two CA-C proteins as a function of separation and amino acid sequence in the interfacial region. The simulations demonstrate that CA-C protein-protein interactions sit at the edge of a dewetting transition and that this mesoscopic manifestation of the underlying liquid-vapor phase transition can be readily manipulated by biology or protein engineering to significantly affect association behavior. While the wild type protein remains wet until contact, we identify a set of in silico mutations, in which three hydrophilic amino acids are replaced with nonpolar residues, that leads to dewetting prior to association. The existence of dewetting depends on the size and relative locations of substituted residues separated by nm length scales, indicating long range cooperativity and a sensitivity to surface topography. These observations identify important details which are missing from descriptions of protein association based on buried hydrophobic surface area.

  7. Protein dynamics at Eph receptor-ligand interfaces as revealed by crystallography, NMR and MD simulations

    International Nuclear Information System (INIS)

    The role of dynamics in protein functions including signal transduction is just starting to be deciphered. Eph receptors with 16 members divided into A- and B- subclasses are respectively activated by 9 A- and B-ephrin ligands. EphA4 is the only receptor capable of binding to all 9 ephrins and small molecules with overlapped interfaces. We first determined the structures of the EphA4 ligand binding domain (LBD) in two crystals of P1 space group. Noticeably, 8 EphA4 molecules were found in one asymmetric unit and consequently from two crystals we obtained 16 structures, which show significant conformational variations over the functionally critical A-C, D-E, G-H and J-K loops. The 16 new structures, together with previous 9 ones, can be categorized into two groups: closed and open forms which resemble the uncomplexed and complexed structures of the EphA4 LBD respectively. To assess whether the conformational diversity over the loops primarily results from the intrinsic dynamics, we initiated 30-ns molecular dynamics (MD) simulations for both closed and open forms. The results indicate that the loops do have much higher intrinsic dynamics, which is further unravelled by NMR H/D exchange experiments. During simulations, the open form has the RMS deviations slightly larger than those of the closed one, suggesting the open form may be less stable in the absence of external contacts. Furthermore, no obvious exchange between two forms is observed within 30 ns, implying that they are dynamically separated. Our study provides the first experimental and computational result revealing that the intrinsic dynamics are most likely underlying the conformational diversity observed for the EphA4 LBD loops mediating the binding affinity and specificity. Interestingly, the open conformation of the EphA4 LBD is slightly unstable in the absence of it natural ligand ephrins, implying that the conformational transition from the closed to open has to be driven by the high

  8. Label-Free Quantitative Mass Spectrometry Reveals a Panel of Differentially Expressed Proteins in Colorectal Cancer

    OpenAIRE

    2015-01-01

    To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and ...

  9. Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling

    OpenAIRE

    Williams, Christopher C.; Jan, Calvin H.; Weissman, Jonathan S.

    2014-01-01

    Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively measure translation at the mitochondrial surface in yeast. Most inner membrane proteins were co-translationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of protein...

  10. Quantitative proteomic analysis of mice corneal tissues reveals angiogenesis-related proteins involved in corneal neovascularization.

    Science.gov (United States)

    Shen, Minqian; Tao, Yimin; Feng, Yifan; Liu, Xing; Yuan, Fei; Zhou, Hu

    2016-07-01

    Corneal neovascularization (CNV) was induced in Balb/c mice by alkali burns in the central area of the cornea with a diameter of 2.5mm. After fourteen days, the cornea from one eye was collected for histological staining for CNV examination, while the cornea from the other eye of the same mouse was harvested for proteomic analysis. The label-free quantitative proteomic approach was applied to analyze five normal corneal tissues (normal group mice n=5) and five corresponding neovascularized corneal tissues (model group mice n=5). A total of 2124 proteins were identified, and 1682 proteins were quantified from these corneal tissues. Among these quantified proteins, 290 proteins were significantly changed between normal and alkali burned corneal tissues. Of these significantly changed proteins, 35 were reported or predicted as angiogenesis-related proteins. Then, these 35 proteins were analyzed using Ingenuity Pathway Analysis Software, resulting in 26 proteins enriched and connected to each other in the protein-protein interaction network, such as Lcn-2, αB-crystallin and Serpinf1 (PEDF). These three significantly changed proteins were selected for further Western blotting validation. Consistent with the quantitative proteomic results, Western blotting showed that Lcn-2 and αB-crystallin were significantly up-regulated in CNV model, while PEDF was down-regulated. This study provided increased understanding of angiogenesis-related proteins involved in corneal vascular development, which will be useful in the ophthalmic clinic of specifically target angiogenesis. PMID:27049463

  11. Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications*

    OpenAIRE

    Halim, Adnan; Carlsson, Michael C.; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y.; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L.; Wandall, Hans H.

    2014-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited.

  12. A P4-ATPase protein interaction network reveals a link between aminophospholipid transport and phosphoinositide metabolism

    NARCIS (Netherlands)

    Puts, C.F.; Lenoir, G.F.; Krijgsveld, J.; WIlliamson, P.L.; Holthuis, J.C.M.

    2009-01-01

    High-throughput analysis of protein-protein interactions can provide unprecedented insight into how cellular processes are integrated at the molecular level. Yet membrane proteins are often overlooked in these studies owing to their hydrophobic nature and low abundance. Here we used a proteomics-bas

  13. Crystal structure of the Campylobacter jejuni Cj0090 protein reveals a novel variant of the immunoglobulin fold among bacterial lipoproteins.

    Science.gov (United States)

    Paek, Seonghee; Kawai, Fumihiro; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2012-12-01

    Bacterial lipoproteins play an important role in bacterial pathogenesis and physiology. The genome of Campylobacter jejuni, a major foodborn pathogen, is predicted to contain over 20 lipoproteins. However, the functions of the majority of C. jejuni lipoproteins remain unknown. The Cj0090 protein is encoded by a lipoprotein operon composed of cj0089, cj0090, and cj0091. Here, we report the crystal structure of Cj0090 at 1.9 Å resolution, revealing a novel variant of the immunoglobulin fold with β-sandwich architecture. The structure suggests that Cj0090 may be involved in protein-protein interactions, consistent with a possible role for bacterial lipoproteins. PMID:22987763

  14. New insights on the sialidase protein family revealed by a phylogenetic analysis in metazoa.

    Directory of Open Access Journals (Sweden)

    Edoardo Giacopuzzi

    Full Text Available Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. These enzymes modulate the cell sialic acid content and are involved in several cellular processes and pathological conditions. Molecular defects in NEU1 are responsible for sialidosis, an inherited disease characterized by lysosomal storage disorder and neurodegeneration. The studies on the biology of sialic acids and sialyltransferases, the anabolic counterparts of sialidases, have revealed a complex picture with more than 50 sialic acid variants selectively present in the different branches of the tree of life. The gain/loss of specific sialoconjugates have been proposed as key events in the evolution of deuterostomes and Homo sapiens, as well as in the host-pathogen interactions. To date, less attention has been paid to the evolution of sialidases. Thus we have conducted a survey on the state of the sialidase family in metazoan. Using an in silico approach, we identified and characterized sialidase orthologs from 21 different organisms distributed among the evolutionary tree: Metazoa relative (Monosiga brevicollis, early Deuterostomia, precursor of Chordata and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals. We were able to reconstruct the evolution of the sialidase protein family from the ancestral sialidase NEU1 and identify a new form of the enzyme, NEU5, representing an intermediate step in the evolution leading to the modern NEU3, NEU4 and NEU2. Our study provides new insights on the mechanisms that shaped the substrate specificity and other peculiar properties of the modern mammalian sialidases. Moreover, we further confirm findings on the catalytic residues and identified enzyme loop portions that behave as rapidly diverging regions and may

  15. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

    Science.gov (United States)

    Li, Renfeng; Liao, Gangling; Nirujogi, Raja Sekhar; Pinto, Sneha M; Shaw, Patrick G; Huang, Tai-Chung; Wan, Jun; Qian, Jiang; Gowda, Harsha; Wu, Xinyan; Lv, Dong-Wen; Zhang, Kun; Manda, Srikanth S; Pandey, Akhilesh; Hayward, S Diane

    2015-12-01

    Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in

  16. Identification of the interactome between fish plasma proteins and Edwardsiella tarda reveals tissue-specific strategies against bacterial infection.

    Science.gov (United States)

    Li, Hui; Huang, Xiaoyan; Zeng, Zaohai; Peng, Xuan-Xian; Peng, Bo

    2016-09-01

    Elucidating the complex pathogen-host interaction is essential for a comprehensive understanding of how these remarkable agents invade their hosts and how the hosts defend against these invaders. During the infection, pathogens interact intensively with host to enable their survival, which can be revealed through their interactome. Edwardsiella tarda is a Gram-negative bacterial pathogen causing huge economic loss in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. E. tarda is an ideal model for host-pathogen investigation as it infects fish in three distinct steps: entering the host, circulating through the blood and establishing infection. We adopted a previous established proteomic approach that inactivated E. tarda cells and covalent crosslink fish plasma proteins were used to capture plasma proteins and bacterial outer membrane proteins, respectively. By the combinatorial use of proteomic and biochemical approaches, six plasma proteins and seven outer membrane proteins (OMPs) were identified. Interactions among these proteins were validated with protein-array, far-Western blotting and co-immunoprecipitation. At last, seventeen plasma protein-bacteria protein-protein interaction were confirmed to be involved in the interaction network, forming a complex interactome. Compared to our previous results, different host proteins were detected, whereas some of the bacterial proteins were similar, which indicates that hosts adopt tissue-specific strategies to cope with the same pathogen during infection. Thus, our results provide a robust demonstration of both bacterial initiators and host receptors or interacting proteins to further explore infection and anti-infective mechanisms between hosts and microbes. PMID:27458055

  17. A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum.

    Directory of Open Access Journals (Sweden)

    Stefanie Wolfram

    Full Text Available Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways.

  18. The spider hemolymph clot proteome reveals high concentrations of hemocyanin and von Willebrand factor-like proteins.

    Science.gov (United States)

    Sanggaard, Kristian W; Dyrlund, Thomas F; Bechsgaard, Jesper S; Scavenius, Carsten; Wang, Tobias; Bilde, Trine; Enghild, Jan J

    2016-02-01

    Arthropods include chelicerates, crustaceans, and insects that all have open circulation systems and thus require different properties of their coagulation system than vertebrates. Although the clotting reaction in the chelicerate horseshoe crab (Family: Limulidae) has been described in details, the overall protein composition of the resulting clot has not been analyzed for any of the chelicerates. The largest class among the chelicerates is the arachnids, which includes spiders, ticks, mites, and scorpions. Here, we use a mass spectrometry-based approach to characterize the spider hemolymph clot proteome from the Brazilian whiteknee tarantula, Acanthoscurria geniculata. We focused on the insoluble part of the clot and demonstrated high concentrations of proteins homologous to the hemostasis-related and multimerization-prone von Willebrand factor. These proteins, which include hemolectins and vitellogenin homologous, were previously identified as essential components of the hemolymph clot in crustaceans and insects. Their presence in the spider hemolymph clot suggests that the origin of these proteins' function in coagulation predates the split between chelicerates and mandibulata. The clot proteome reveals that the major proteinaceous component is the oxygen-transporting and phenoloxidase-displaying abundant hemolymph protein hemocyanin, suggesting that this protein also plays a role in clot biology. Furthermore, quantification of the peptidome after coagulation revealed the simultaneous activation of both the innate immune system and the coagulation system. In general, many of the identified clot-proteins are related to the innate immune system, and our results support the previously suggested crosstalk between immunity and coagulation in arthropods. PMID:26621385

  19. Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.

    Science.gov (United States)

    Rocheleau, Gail; Petrillo, Jessica; Guogas, Laura; Gehrke, Lee

    2004-08-01

    The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity. PMID:15254175

  20. The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

    Directory of Open Access Journals (Sweden)

    Maheswara Reddy Emani

    2015-03-01

    Full Text Available The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

  1. Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins.

    Science.gov (United States)

    Narumi, Ryohei; Shimizu, Yoshihiro; Ukai-Tadenuma, Maki; Ode, Koji L; Kanda, Genki N; Shinohara, Yuta; Sato, Aya; Matsumoto, Katsuhiko; Ueda, Hiroki R

    2016-06-14

    Absolute values of protein expression levels in cells are crucial information for understanding cellular biological systems. Precise quantification of proteins can be achieved by liquid chromatography (LC)-mass spectrometry (MS) analysis of enzymatic digests of proteins in the presence of isotope-labeled internal standards. Thus, development of a simple and easy way for the preparation of internal standards is advantageous for the analyses of multiple target proteins, which will allow systems-level studies. Here we describe a method, termed MS-based Quantification By isotope-labeled Cell-free products (MS-QBiC), which provides the simple and high-throughput preparation of internal standards by using a reconstituted cell-free protein synthesis system, and thereby facilitates both multiplexed and sensitive quantification of absolute amounts of target proteins. This method was applied to a systems-level dynamic analysis of mammalian circadian clock proteins, which consist of transcription factors and protein kinases that govern central and peripheral circadian clocks in mammals. Sixteen proteins from 20 selected circadian clock proteins were successfully quantified from mouse liver over a 24-h time series, and 14 proteins had circadian variations. Quantified values were applied to detect internal body time using a previously developed molecular timetable method. The analyses showed that single time-point data from wild-type mice can predict the endogenous state of the circadian clock, whereas data from clock mutant mice are not applicable because of the disappearance of circadian variation. PMID:27247408

  2. Proteomic Analysis Reveals the Deregulation of Inflammation-Related Proteins in Acupuncture-Treated Rats with Asthma Onset

    Directory of Open Access Journals (Sweden)

    Yu-Dong Xu

    2012-01-01

    Full Text Available Although the beneficial effects of acupuncture in asthma treatment have been well documented, little is known regarding the biological basis of this treatment. Changes in the lung proteome of acupuncture-treated rats with asthma onset were comparatively analyzed using a two-dimensional gel electrophoresis (2DE and mass-spectrometry- (MS- based proteomic approach. Acupuncture on specific acupuncture points appeared to improve respiratory function and reduce the total number of leukocytes and eosinophils in bronchoalveolar lavage fluid in OVA-induced asthma onset. Image analysis of 2DE gels revealed 32 differentially expressed acupuncture-specific protein spots in asthma onset; 30 of which were successfully identified as 28 unique proteins using LC-MS/MS. Bioinformatic analyses indicated that these altered proteins are most likely involved in inflammation-related biological functions, and the functional associations of these proteins result in an inflammation signaling pathway. Acupuncture regulates the pathway at different levels by regulating several key nodal proteins, including downregulating of proinflammatory proteins (e.g., S100A8, RAGE, and S100A11 and upregulating of anti-inflammatory proteins (e.g., CC10, ANXA5, and sRAGE. These deregulated inflammation-related proteins may mediate, at least in part, the antiasthmatic effect of acupuncture. Further functional investigation of these acupuncture-specific effector proteins could identify new drug candidates for the prophylaxis and treatment of asthma.

  3. Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions

    OpenAIRE

    Beld, Joris; Jillian L Blatti; Behnke, Craig; Mendez, Michael; Burkart, Michael D.

    2013-01-01

    The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the p...

  4. Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells

    OpenAIRE

    Barhoom, Sima; Farrell, Ian; Shai, Ben; Dahary, Dvir; Cooperman, Barry S.; Smilansky, Zeev; Elroy-Stein, Orna; Ehrlich, Marcelo

    2013-01-01

    The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (...

  5. Simulations of HIV capsid protein dimerization reveal the effect of chemistry and topography on the mechanism of hydrophobic protein association

    CERN Document Server

    Yu, Naiyin

    2015-01-01

    Recent work has shown that the hydrophobic protein surfaces in aqueous solution sit near a drying transition. The tendency for these surfaces to expel water from their vicinity leads to self assembly of macromolecular complexes. In this article we show with a realistic model for a biologically pertinent system how this phenomenon appears at the molecular level. We focus on the association of the C-terminal domain (CA-C) of the human immunodeficiency virus (HIV) capsid protein. By combining all-atom simulations with specialized sampling techniques we measure the water density distribution during the approach of two CA-C proteins as a function of separation and amino acid sequence in the interfacial region. The simulations demonstrate that CA-C protein-protein interactions sit at the edge of a dewetting transition and that this mesoscopic manifestation of the underlying liquid-vapor phase transition can be readily manipulated by biology or protein engineering to significantly affect association behavior. While ...

  6. Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain

    Directory of Open Access Journals (Sweden)

    Suzan-Monti Marie

    2009-05-01

    Full Text Available Abstract Background Acanthamoebae polyphaga Mimivirus (APM is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. However this protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein. Results This study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family. Conclusion The characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.

  7. Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

    Science.gov (United States)

    Miclăuş, Teodora; Beer, Christiane; Chevallier, Jacques; Scavenius, Carsten; Bochenkov, Vladimir E.; Enghild, Jan J.; Sutherland, Duncan S.

    2016-06-01

    Proteins adsorbing at nanoparticles have been proposed as critical toxicity mediators and are included in ongoing efforts to develop predictive tools for safety assessment. Strongly attached proteins can be isolated, identified and correlated to changes in nanoparticle state, cellular association or toxicity. Weakly attached, rapidly exchanging proteins are also present at nanoparticles, but are difficult to isolate and have hardly been examined. Here we study rapidly exchanging proteins and show for the first time that they have a strong modulatory effect on the biotransformation of silver nanoparticles. Released silver ions, known for their role in particle toxicity, are found to be trapped as silver sulphide nanocrystals within the protein corona at silver nanoparticles in serum-containing cell culture media. The strongly attached corona acts as a site for sulphidation, while the weakly attached proteins reduce nanocrystal formation in a serum-concentration-dependent manner. Sulphidation results in decreased toxicity of Ag NPs.

  8. Combinations of protein-chemical complex structures reveal new targets for established drugs.

    OpenAIRE

    Kalinina, Olga V.; Oliver Wichmann; Gordana Apic; Russell, Robert B.

    2011-01-01

    Biological networks are powerful tools for predicting undocumented relationships between molecules. The underlying principle is that existing interactions between molecules can be used to predict new interactions. Here we use this principle to suggest new protein-chemical interactions via the network derived from three-dimensional structures. For pairs of proteins sharing a common ligand, we use protein and chemical superimpositions combined with fast structural compatibility screens to predi...

  9. A genome wide study in fission yeast reveals nine PPR proteins that regulate mitochondrial gene expression

    OpenAIRE

    Kühl, Inge; Dujeancourt, Laurent; Gaisne, Mauricette; Herbert, Christopher J.; Bonnefoy, Nathalie

    2011-01-01

    Pentatricopeptide repeat (PPR) proteins are particularly numerous in plant mitochondria and chloroplasts, where they are involved in different steps of RNA metabolism, probably due to the repeated 35 amino acid PPR motifs that are thought to mediate interactions with RNA. In non-photosynthetic eukaryotes only a handful of PPR proteins exist, for example the human LRPPRC, which is involved in a mitochondrial disease. We have conducted a systematic study of the PPR proteins in the fission yeast...

  10. Crystal Structure of Menin Reveals Binding Site for Mixed Lineage Leukemia (MLL) Protein

    Energy Technology Data Exchange (ETDEWEB)

    Murai, Marcelo J.; Chruszcz, Maksymilian; Reddy, Gireesh; Grembecka, Jolanta; Cierpicki, Tomasz (Michigan); (UV)

    2014-10-02

    Menin is a tumor suppressor protein that is encoded by the MEN1 (multiple endocrine neoplasia 1) gene and controls cell growth in endocrine tissues. Importantly, menin also serves as a critical oncogenic cofactor of MLL (mixed lineage leukemia) fusion proteins in acute leukemias. Direct association of menin with MLL fusion proteins is required for MLL fusion protein-mediated leukemogenesis in vivo, and this interaction has been validated as a new potential therapeutic target for development of novel anti-leukemia agents. Here, we report the first crystal structure of menin homolog from Nematostella vectensis. Due to a very high sequence similarity, the Nematostella menin is a close homolog of human menin, and these two proteins likely have very similar structures. Menin is predominantly an {alpha}-helical protein with the protein core comprising three tetratricopeptide motifs that are flanked by two {alpha}-helical bundles and covered by a {beta}-sheet motif. A very interesting feature of menin structure is the presence of a large central cavity that is highly conserved between Nematostella and human menin. By employing site-directed mutagenesis, we have demonstrated that this cavity constitutes the binding site for MLL. Our data provide a structural basis for understanding the role of menin as a tumor suppressor protein and as an oncogenic co-factor of MLL fusion proteins. It also provides essential structural information for development of inhibitors targeting the menin-MLL interaction as a novel therapeutic strategy in MLL-related leukemias.

  11. Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

    Science.gov (United States)

    Ouidir, Tassadit; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2015-07-01

    Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology. PMID:25900529

  12. Massively parallel sampling of lattice proteins reveals foundations of thermal adaptation

    Science.gov (United States)

    Venev, Sergey V.; Zeldovich, Konstantin B.

    2015-08-01

    Evolution of proteins in bacteria and archaea living in different conditions leads to significant correlations between amino acid usage and environmental temperature. The origins of these correlations are poorly understood, and an important question of protein theory, physics-based prediction of types of amino acids overrepresented in highly thermostable proteins, remains largely unsolved. Here, we extend the random energy model of protein folding by weighting the interaction energies of amino acids by their frequencies in protein sequences and predict the energy gap of proteins designed to fold well at elevated temperatures. To test the model, we present a novel scalable algorithm for simultaneous energy calculation for many sequences in many structures, targeting massively parallel computing architectures such as graphics processing unit. The energy calculation is performed by multiplying two matrices, one representing the complete set of sequences, and the other describing the contact maps of all structural templates. An implementation of the algorithm for the CUDA platform is available at http://www.github.com/kzeldovich/galeprot and calculates protein folding energies over 250 times faster than a single central processing unit. Analysis of amino acid usage in 64-mer cubic lattice proteins designed to fold well at different temperatures demonstrates an excellent agreement between theoretical and simulated values of energy gap. The theoretical predictions of temperature trends of amino acid frequencies are significantly correlated with bioinformatics data on 191 bacteria and archaea, and highlight protein folding constraints as a fundamental selection pressure during thermal adaptation in biological evolution.

  13. Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells.

    Science.gov (United States)

    Barhoom, Sima; Farrell, Ian; Shai, Ben; Dahary, Dvir; Cooperman, Barry S; Smilansky, Zeev; Elroy-Stein, Orna; Ehrlich, Marcelo

    2013-10-01

    The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNA(Ile)UAU-generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA-mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein. PMID:23965304

  14. Localization of Seed Oil Body Proteins in Tobacco Protoplasts Reveals Specific Mechanisms of Protein Targeting to Leaf Lipid Droplets

    Institute of Scientific and Technical Information of China (English)

    Stefania De Domenico; Stefania Bonsegna; Marcello Salvatore Lenucci; Palmiro Poltronieri; Gian Pietro Di Sansebastiano; Angelo Santino

    2011-01-01

    Oleosin,caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies.In the present work,the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy.Our results indicated clear differences in the endocellular localization of the three proteins.Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets (LD),whereas steroleosin,characterized by an N-terminal oil body anchoring domain,was mainly retained in the endoplasmic reticulum (ER).Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosingreen fluorescent protein (GFP) peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein (BiP),were recovered.Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered.Finally,only oleosinand caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids.Taken together,our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.

  15. Molecular shape and binding force of Mycoplasma mobile's leg protein Gli349 revealed by an AFM study

    Energy Technology Data Exchange (ETDEWEB)

    Lesoil, Charles [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsutacho 4259, Midori-ku, Yokohama 226-8501 (Japan); Nonaka, Takahiro [Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585 (Japan); Sekiguchi, Hiroshi; Osada, Toshiya [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsutacho 4259, Midori-ku, Yokohama 226-8501 (Japan); Miyata, Makoto [Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585 (Japan); Afrin, Rehana [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsutacho 4259, Midori-ku, Yokohama 226-8501 (Japan); Biofrontier Center, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsutacho 4259, Midori-ku, Yokohama 226-8501 (Japan); Ikai, Atsushi, E-mail: ikai.a.aa@m.titech.ac.jp [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsutacho 4259, Midori-ku, Yokohama 226-8501 (Japan)

    2010-01-15

    Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349 kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the 'leg' of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70 pN. These findings strongly support the idea that Gli349 is the 'leg' protein of M. mobile, responsible for binding and also most probably force generation during gliding motility.

  16. Molecular shape and binding force of Mycoplasma mobile's leg protein Gli349 revealed by an AFM study

    International Nuclear Information System (INIS)

    Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349 kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the 'leg' of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70 pN. These findings strongly support the idea that Gli349 is the 'leg' protein of M. mobile, responsible for binding and also most probably force generation during gliding motility.

  17. Comparative proteomic analysis of the silkworm middle silk gland reveals the importance of ribosome biogenesis in silk protein production.

    Science.gov (United States)

    Li, Jian-ying; Ye, Lu-peng; Che, Jia-qian; Song, Jia; You, Zheng-ying; Yun, Ki-chan; Wang, Shao-hua; Zhong, Bo-xiong

    2015-08-01

    The silkworm middle silk gland (MSG) is the sericin synthesis and secretion unique sub-organ. The molecular mechanisms of regulating MSG protein synthesis are largely unknown. Here, we performed shotgun proteomic analysis on the three MSG subsections: the anterior (MSG-A), middle (MSG-M), and posterior (MSG-P) regions. The results showed that more strongly expressed proteins in the MSG-A were involved in multiple processes, such as silk gland development and silk protein protection. The proteins that were highly expressed in the MSG-M were enriched in the ribosome pathway. MSG-P proteins with stronger expression were mainly involved in the oxidative phosphorylation and citrate cycle pathways. These results suggest that the MSG-M is the most active region in the sericin synthesis. Furthermore, comparing the proteome of the MSG with the posterior silk gland (PSG) revealed that the specific and highly expressed proteins in the MSG were primarily involved in the ribosome and aminoacyl-tRNA biosynthesis pathways. These results indicate that silk protein synthesis is much more active as a result of the enhancement of translation-related pathways in the MSG. These results also suggest that enhancing ribosome biogenesis is important to the efficient synthesis of silk proteins. PMID:26051239

  18. Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

    International Nuclear Information System (INIS)

    Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development

  19. Proteomic and Functional Analyses Reveal MAPK1 Regulates Milk Protein Synthesis

    Directory of Open Access Journals (Sweden)

    Xue-Jun Gao

    2012-12-01

    Full Text Available L-Lysine (L-Lys is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs. The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC. The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

  20. Dynamic changes in protein functional linkage networks revealed by integration with gene expression data.

    Directory of Open Access Journals (Sweden)

    Shubhada R Hegde

    2008-11-01

    Full Text Available Response of cells to changing environmental conditions is governed by the dynamics of intricate biomolecular interactions. It may be reasonable to assume, proteins being the dominant macromolecules that carry out routine cellular functions, that understanding the dynamics of protein:protein interactions might yield useful insights into the cellular responses. The large-scale protein interaction data sets are, however, unable to capture the changes in the profile of protein:protein interactions. In order to understand how these interactions change dynamically, we have constructed conditional protein linkages for Escherichia coli by integrating functional linkages and gene expression information. As a case study, we have chosen to analyze UV exposure in wild-type and SOS deficient E. coli at 20 minutes post irradiation. The conditional networks exhibit similar topological properties. Although the global topological properties of the networks are similar, many subtle local changes are observed, which are suggestive of the cellular response to the perturbations. Some such changes correspond to differences in the path lengths among the nodes of carbohydrate metabolism correlating with its loss in efficiency in the UV treated cells. Similarly, expression of hubs under unique conditions reflects the importance of these genes. Various centrality measures applied to the networks indicate increased importance for replication, repair, and other stress proteins for the cells under UV treatment, as anticipated. We thus propose a novel approach for studying an organism at the systems level by integrating genome-wide functional linkages and the gene expression data.

  1. Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis.

    Science.gov (United States)

    Küberl, Andreas; Fränzel, Benjamin; Eggeling, Lothar; Polen, Tino; Wolters, Dirk Andreas; Bott, Michael

    2014-06-01

    In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome-lacking non-pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni(2+)-chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI-TOF-MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins. PMID:24737727

  2. Plasma proteome profiling of atherosclerotic disease manifestations reveals elevated levels of the cytoskeletal protein vinculin

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Larsen, Martin Røssel; Mickley, Hans; Saaby, Lotte; Diederichsen, Axel Cosmus Pyndt; Lambrechtsen, Jess; Rasmussen, Lars M; Overgaard, Martin

    2014-01-01

    atherosclerotic diseases, and 4) individuals with an acute coronary syndrome. Immunoassays and SRM-MS were used for single patient verification of candidate proteins. Proteins involved in cardiovascular diseases i.e. serum amyloid protein A (SAA), C-reactive protein (CRP), and apolipoprotein(a) [apo(a)] displayed...... identify proteins with altered concentrations in plasma samples from four groups: 1) Individuals without cardiovascular symptoms and without the presence of coronary calcium, 2) individuals without cardiovascular symptoms, but with high amounts of coronary calcium, 3) individuals operated because of......Atherosclerosis is a chronic disease of the arterial wall that is recognized as the leading cause of mortality and morbidity worldwide. There is an eminent need for better biomarkers that can aid in patient care before the onset of the first cardiovascular event. We used quantitative proteomics to...

  3. Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential.

    Directory of Open Access Journals (Sweden)

    Stéphane Bellafiore

    2008-10-01

    Full Text Available The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins. Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth. Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi, suggesting a common parasitic behavior and a possible

  4. A new tool in C. elegans reveals changes in secretory protein metabolism in ire-1-deficient animals.

    Science.gov (United States)

    Safra, Modi; Henis-Korenblit, Sivan

    2014-01-01

    We recently showed that the ire-1/xbp-1 arm of the UPR plays a crucial role in maintaining basic endoplasmic reticulum (ER) functions required for the metabolism of secreted proteins even during unstressed growth conditions. During these studies we realized that although C. elegans is a powerful system to study the genetics of many cellular processes; it lacks effective tools for tracking the metabolism of secreted proteins at the cell and organism levels. Here, we outline how genetic manipulations and expression analysis of a DAF-28::GFP translational fusion transgene can be combined to infer different steps in the life cycle of secretory proteins. We demonstrate how we have used this tool to reveal folding defects, clearance defects, and secretion defects in ire-1 and xbp-1 mutants. We believe that further studies using this tool will deepen the understanding of secretory protein metabolism. PMID:25191629

  5. Quantitative Proteomics Analysis Reveals the Min System of Escherichia coli Modulates Reversible Protein Association with the Inner Membrane.

    Science.gov (United States)

    Lee, Hsiao-Lin; Chiang, I-Chen; Liang, Suh-Yuen; Lee, Der-Yen; Chang, Geen-Dong; Wang, Kwan-Yu; Lin, Shu-Yu; Shih, Yu-Ling

    2016-05-01

    The Min system of Escherichia coli mediates placement of the division septum at the midcell. It oscillates from pole to pole to establish a concentration gradient of the division inhibition that is high at the poles but low at the midcell; the cell middle thereby becomes the most favorable site for division. Although Min oscillation is well studied from molecular and biophysical perspectives, it is still an enigma as to whether such a continuous, energy-consuming, and organized movement of the Min proteins would affect cellular processes other than the division site selection. To tackle this question, we compared the inner membrane proteome of the wild-type and Δmin strains using a quantitative approach. Forty proteins that showed differential abundance on the inner membrane of the mutant cells were identified and defined as proteins of interest (POIs). More than half of the POIs were peripheral membrane proteins, suggesting that the Min system affects mainly reversible protein association with the inner membrane. In addition, 6 out of 10 selected POIs directly interacted with at least one of the Min proteins, confirming the correlation between POIs and the Min system.Further analysis revealed a functional relationship between metabolism and the Min system. Metabolic enzymes accounted for 45% of the POIs, and there was a change of metabolites in the related reactions. We hypothesize that the Min system could alter the membrane location of proteins to modulate their enzymatic activity. Thus, the metabolic modulation in the Δmin mutant is likely an adaptive phenotype in cells of abnormal size and chromosome number due to an imbalanced abundance of proteins on the inner membrane. Taken together, the current work reports novel interactions of the Min system and reveals a global physiological impact of the Min system in addition to the division site placement. PMID:26889046

  6. Proteomic analysis of pure human airway gland mucus reveals a large component of protective proteins.

    Directory of Open Access Journals (Sweden)

    Nam Soo Joo

    Full Text Available Airway submucosal glands contribute to innate immunity and protect the lungs by secreting mucus, which is required for mucociliary clearance and which also contains antimicrobial, anti-inflammatory, anti-proteolytic and anti-oxidant proteins. We stimulated glands in tracheal trimmings from three lung donors and collected droplets of uncontaminated mucus as they formed at the gland orifices under an oil layer. We analyzed the mucus using liquid chromatography-tandem mass spectrometry (LC-MS/MS. Analysis identified 5486 peptides and 441 proteins from across the 3 samples (269-319 proteins per subject. We focused on 269 proteins common to at least 2 0f 3 subjects, of which 102 (38% had protective or innate immunity functions. While many of these have long been known to play such roles, for many others their cellular protective functions have only recently been appreciated in addition to their well-studied biologic functions (e.g. annexins, apolipoproteins, gelsolin, hemoglobin, histones, keratins, and lumican. A minority of the identified proteins are known to be secreted via conventional exocytosis, suggesting that glandular secretion occurs via multiple mechanisms. Two of the observed protective proteins, major vault protein and prohibitin, have not been observed in fluid from human epithelial cultures or in fluid from nasal or bronchoalveolar lavage. Further proteomic analysis of pure gland mucus may help clarify how healthy airways maintain a sterile environment.

  7. Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications

    DEFF Research Database (Denmark)

    Halim, Adnan; Carlsson, Michael C; Mathiesen, Caroline Benedicte K;

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post......-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM...

  8. Label-free quantitative phosphoproteomic analysis reveals differentially regulated proteins and pathway in PRRSV-infected pulmonary alveolar macrophages.

    Science.gov (United States)

    Luo, Rui; Fang, Liurong; Jin, Hui; Wang, Dang; An, Kang; Xu, Ningzhi; Chen, Huanchun; Xiao, Shaobo

    2014-03-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine worldwide and causes significant economic losses. Through regulating the host proteins phosphorylation, PRRSV was found to manipulate the activities of several signaling molecules to regulate innate immune responses. However, the role of protein phosphorylation during PRRSV infection and the signal pathways responsible for it are relatively unknown. Here liquid chromatography-tandem mass spectrometry for label-free quantitative phosphoproteomics was applied to systematically investigate the global phosphorylation events in PRRSV-infected pulmonary alveolar macrophages. In total, we identified 2125 unique phosphosites, of which the phosphorylation level of 292 phosphosites on 242 proteins and 373 phosphosites on 249 proteins was significantly altered at 12 and 36 h pi, respectively. The phosphoproteomics data were analyzed using ingenuity pathways analysis to identify defined canonical pathways and functional networks. Pathway analysis revealed that PRRSV-induced inflammatory cytokines production was probably due to the activation of mitogen-activated protein kinase and NF-κB signal pathway, which were regulated by several protein kinases during virus infection. Interacting network analysis indicated that altered phosphoproteins were involved in cellular assembly and organization, protein synthesis, molecular transport, and signal transduction in PRRSV infected cells. These pathways and functional networks analysis could provide direct insights into the biological significance of phosphorylation events modulated by PRRSV and may help us elucidate the pathogenic mechanisms of PRRSV infection. PMID:24533505

  9. Proteomic analysis of the mature Brassica stigma reveals proteins with diverse roles in vegetative and reproductive development.

    Science.gov (United States)

    Nazemof, Nazila; Couroux, Philippe; Xing, Tim; Robert, Laurian S

    2016-09-01

    The stigma, the specialized apex of the Brassicaceae gynoecium, plays a role in pollen capture, discrimination, hydration, germination, and guidance. Despite this crucial role in reproduction, the global proteome underlying Brassicaceae stigma development and function remains largely unknown. As a contribution towards the characterization of the Brassicaceae dry stigma global proteome, more than 2500 Brassica napus mature stigma proteins were identified using three different gel-based proteomics approaches. Most stigma proteins participated in Metabolic Processes, Responses to Stimulus or Stress, Cellular or Developmental Processes, and Transport. The stigma was found to express a wide variety of proteins with demonstrated roles in cellular and organ development including proteins known to be involved in cellular expansion and morphogenesis, embryo development, as well as gynoecium and stigma development. Comparisons to a corresponding proteome from a very morphologically different Poaceae dry stigma showed a very similar distribution of proteins among different functional categories, but also revealed evident distinctions in protein composition especially in glucosinolate and carotenoid metabolism, photosynthesis, and self-incompatibility. To our knowledge, this study reports the largest Brassicaceae stigma protein dataset described to date. PMID:27457983

  10. Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes.

    Directory of Open Access Journals (Sweden)

    Chang-Cai Liu

    Full Text Available BACKGROUND: The N-terminal protein processing mechanism (NPM including N-terminal Met excision (NME and N-terminal acetylation (N(α-acetylation represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To reveal the NPM in poplar, we investigated the N(α-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α-acetylated proteins. Most proteins (47, >81% are subjected to N(α-acetylation following the N-terminal removal of Met, indicating that N(α-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α-acetylation (NPM to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs and N-terminal acetyltransferase (Nat enzymes in poplar. The N(α-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. CONCLUSIONS/SIGNIFICANCE: This study represents the first extensive investigation of N(α-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α-acetylation of proteins in poplar.

  11. Pathogen protection and virulence: Dark side of fungal membrane protein revealed

    OpenAIRE

    Whyte, Barry James

    2009-01-01

    Researchers at the Virginia Bioinformatics Institute at Virginia Tech and Montana State University have discovered a fungal protein that plays a key role in causing disease in plants and animals and which also shields the pathogen from oxidative stress.

  12. Targeted Chemical Wedges Reveal the Role of Allosteric DNA Modulation in Protein — DNA Assembly

    OpenAIRE

    Moretti, Rocco; Donato, Leslie J.; Brezinski, Mary L.; Stafford, Ryan L.; Hoff, Helena; Thorson, Jon S.; Dervan, Peter B.; Ansari, Aseem Z.

    2008-01-01

    The cooperative assembly of multiprotein complexes results from allosteric modulations of DNA structure as well as direct intermolecular contacts between proteins. Such cooperative binding plays a critical role in imparting exquisite sequence specificity on the homeobox transcription factor (Hox) family of developmental transcription factors. A well-characterized example includes the interaction of Hox proteins with extradenticle (Exd), a highly conserved DNA binding transcription factor. Alt...

  13. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn

    OpenAIRE

    Yongbin Dong; Qilei Wang; Long Zhang; Chunguang Du; Wenwei Xiong; Xinjian Chen; Fei Deng; Zhiyan Ma; Dahe Qiao; Chunhui Hu; Yangliu Ren; Yuling Li

    2015-01-01

    The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses...

  14. QUANTUM DOT SINGLE MOLECULE TRACKING REVEALS A WIDE RANGE OF DIFFUSIVE MOTIONS OF MEMBRANE TRANSPORT PROTEINS

    OpenAIRE

    Crane, Jonathan M.; Haggie, Peter M.; Verkman, A S

    2009-01-01

    Single particle tracking (SPT) provides information about the microscopic motions of individual particles in live cells. We applied SPT to study the diffusion of membrane transport proteins in cell plasma membranes in which individual proteins are labeled with quantum dots at engineered extracellular epitopes. Software was created to deduce particle diffusive modes from quantum dot trajectories. SPT of aquaporin (AQP) water channels and cystic fibrosis transmembrane conductance regulator (CFT...

  15. X-Ray Structures of Native HIV-1 Capsid Protein Reveal Conformational Variability

    OpenAIRE

    Gres, Anna T.; Kirby, Karen A.; KewalRamani, Vineet N.; Tanner, John J.; Pornillos, Owen; Sarafianos, Stefan G.

    2015-01-01

    The detailed molecular interactions between Human Immunodeficiency Virus type 1 (HIV-1) capsid protein (CA) hexamers have been elusive in the context of a native protein. We report crystal structures describing novel interactions between CA monomers related by 6-fold symmetry within a hexamer (intra-hexamer) and by 3-fold and 2-fold symmetry between neighboring hexamers (inter-hexamer). These structures help elucidate how CA builds a hexagonal lattice, the foundation of the mature capsid. Lat...

  16. Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

    OpenAIRE

    la Fuente van Bentem, Sergio de; Anrather, Dorothea; Roitinger, Elisabeth; Djamei, Armin; Hufnagl, Thomas; Barta, Andrea; Csaszar, Edina; Dohnal, Ilse; Lecourieux, David; Hirt, Heribert

    2006-01-01

    Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in...

  17. Structural fragment clustering reveals novel structural and functional motifs in α-helical transmembrane proteins

    Directory of Open Access Journals (Sweden)

    Vassilev Boris

    2010-04-01

    Full Text Available Abstract Background A large proportion of an organism's genome encodes for membrane proteins. Membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. With the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology. Results We introduce a technique called structural fragment clustering, which learns sequential motifs from 3D structural fragments. From over 500,000 fragments, we obtain 213 statistically significant, non-redundant, and novel motifs that are highly specific to α-helical transmembrane proteins. From these 213 motifs, 58 of them were assigned to function and checked in the scientific literature for a biological assessment. Seventy percent of the motifs are found in co-factor, ligand, and ion binding sites, 30% at protein interaction interfaces, and 12% bind specific lipids such as glycerol or cardiolipins. The vast majority of motifs (94% appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. We describe three novel motifs in detail: (1 a dimer interface motif found in voltage-gated chloride channels, (2 a proton transfer motif found in heme-copper oxidases, and (3 a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b. Conclusions Our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. Structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. The sequence motifs can be applied to identify and characterize membrane proteins in novel genomes.

  18. The Population Genomics of Sunflowers and Genomic Determinants of Protein Evolution Revealed by RNAseq

    Directory of Open Access Journals (Sweden)

    Loren H. Rieseberg

    2012-10-01

    Full Text Available Few studies have investigated the causes of evolutionary rate variation among plant nuclear genes, especially in recently diverged species still capable of hybridizing in the wild. The recent advent of Next Generation Sequencing (NGS permits investigation of genome wide rates of protein evolution and the role of selection in generating and maintaining divergence. Here, we use individual whole-transcriptome sequencing (RNAseq to refine our understanding of the population genomics of wild species of sunflowers (Helianthus spp. and the factors that affect rates of protein evolution. We aligned 35 GB of transcriptome sequencing data and identified 433,257 polymorphic sites (SNPs in a reference transcriptome comprising 16,312 genes. Using SNP markers, we identified strong population clustering largely corresponding to the three species analyzed here (Helianthus annuus, H. petiolaris, H. debilis, with one distinct early generation hybrid. Then, we calculated the proportions of adaptive substitution fixed by selection (alpha and identified gene ontology categories with elevated values of alpha. The “response to biotic stimulus” category had the highest mean alpha across the three interspecific comparisons, implying that natural selection imposed by other organisms plays an important role in driving protein evolution in wild sunflowers. Finally, we examined the relationship between protein evolution (dN/dS ratio and several genomic factors predicted to co-vary with protein evolution (gene expression level, divergence and specificity, genetic divergence [FST], and nucleotide diversity pi. We find that variation in rates of protein divergence was correlated with gene expression level and specificity, consistent with results from a broad range of taxa and timescales. This would in turn imply that these factors govern protein evolution both at a microevolutionary and macroevolutionary timescale. Our results contribute to a general understanding of the

  19. Genome-Wide Search for Eliminylating Domains Reveals Novel Function for BLES03-Like Proteins

    OpenAIRE

    Khater, Shradha; Mohanty, Debasisa

    2014-01-01

    Bacterial phosphothreonine lyases catalyze a novel posttranslational modification involving formation of dehydrobutyrine/dehyroalanine by β elimination of the phosphate group of phosphothreonine or phosphoserine residues in their substrate proteins. Though there is experimental evidence for presence of dehydro amino acids in human proteins, no eukaryotic homologs of these lyases have been identified as of today. A comprehensive genome-wide search for identifying phosphothreonine lyase homolog...

  20. NMR spectroscopy reveals unexpected structural variation at the protein-protein interface in MHC class I molecules

    Energy Technology Data Exchange (ETDEWEB)

    Beerbaum, Monika; Ballaschk, Martin; Erdmann, Natalja [Leibniz-Institut fuer Molekulare Pharmakologie (FMP) (Germany); Schnick, Christina [Freie Universitaet Berlin, Institut fuer Immungenetik, Charite-Universitaetsmedizin Berlin (Germany); Diehl, Anne [Leibniz-Institut fuer Molekulare Pharmakologie (FMP) (Germany); Uchanska-Ziegler, Barbara; Ziegler, Andreas [Freie Universitaet Berlin, Institut fuer Immungenetik, Charite-Universitaetsmedizin Berlin (Germany); Schmieder, Peter, E-mail: schmieder@fmp-berlin.de [Leibniz-Institut fuer Molekulare Pharmakologie (FMP) (Germany)

    2013-10-15

    {beta}{sub 2}-Microglobulin ({beta}{sub 2}m) is a small, monomorphic protein non-covalently bound to the heavy chain (HC) in polymorphic major histocompatibility complex (MHC) class I molecules. Given the high evolutionary conservation of structural features of {beta}{sub 2}m in various MHC molecules as shown by X-ray crystallography, {beta}{sub 2}m is often considered as a mere scaffolding protein. Using nuclear magnetic resonance (NMR) spectroscopy, we investigate here whether {beta}{sub 2}m residues at the interface to the HC exhibit changes depending on HC polymorphisms and the peptides bound to the complex in solution. First we show that human {beta}{sub 2}m can effectively be produced in deuterated form using high-cell-density-fermentation and we employ the NMR resonance assignments obtained for triple-labeled {beta}{sub 2}m bound to the HLA-B*27:09 HC to examine the {beta}{sub 2}m-HC interface. We then proceed to compare the resonances of {beta}{sub 2}m in two minimally distinct subtypes, HLA-B*27:09 and HLA-B*27:05, that are differentially associated with the spondyloarthropathy Ankylosing Spondylitis. Each of these subtypes is complexed with four distinct peptides for which structural information is already available. We find that only the resonances at the {beta}{sub 2}m-HC interface show a variation of their chemical shifts between the different complexes. This indicates the existence of an unexpected plasticity that enables {beta}{sub 2}m to accommodate changes that depend on HC polymorphism as well as on the bound peptide through subtle structural variations of the protein-protein interface.

  1. Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants

    OpenAIRE

    Silas P. Rodrigues; José A. Ventura; Aguilar, Clemente; Nakayasu, Ernesto S; Choi, Hyungwon; Sobreira, Tiago J. P.; Nohara, Lilian L.; Wermelinger, Luciana S.; Almeida, Igor C.; Zingali, Russolina B.; Fernandes, Patricia M. B.

    2012-01-01

    Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers’ regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulate...

  2. Cofilin-induced cooperative conformational changes of actin subunits revealed using cofilin-actin fusion protein

    Science.gov (United States)

    Umeki, Nobuhisa; Hirose, Keiko; Uyeda, Taro Q. P.

    2016-01-01

    To investigate cooperative conformational changes of actin filaments induced by cofilin binding, we engineered a fusion protein made of Dictyostelium cofilin and actin. The filaments of the fusion protein were functionally similar to actin filaments bound with cofilin in that they did not bind rhodamine-phalloidin, had quenched fluorescence of pyrene attached to Cys374 and showed enhanced susceptibility of the DNase loop to cleavage by subtilisin. Quantitative analyses of copolymers made of different ratios of the fusion protein and control actin further demonstrated that the fusion protein affects the structure of multiple neighboring actin subunits in copolymers. Based on these and other recent related studies, we propose a mechanism by which conformational changes induced by cofilin binding is propagated unidirectionally to the pointed ends of the filaments, and cofilin clusters grow unidirectionally to the pointed ends following this path. Interestingly, the fusion protein was unable to copolymerize with control actin at pH 6.5 and low ionic strength, suggesting that the structural difference between the actin moiety in the fusion protein and control actin is pH-sensitive. PMID:26842224

  3. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway.

    Science.gov (United States)

    Shi, Tujin; Niepel, Mario; McDermott, Jason E; Gao, Yuqian; Nicora, Carrie D; Chrisler, William B; Markillie, Lye M; Petyuk, Vladislav A; Smith, Richard D; Rodland, Karin D; Sorger, Peter K; Qian, Wei-Jun; Wiley, H Steven

    2016-01-01

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling. PMID:27405981

  4. Comparative proteomics reveals a significant bias toward alternative protein isoforms with conserved structure and function.

    Science.gov (United States)

    Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L

    2012-09-01

    Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of "novel" and "putative" protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and

  5. Small angle neutron scattering contrast variation reveals heterogeneities of interactions in protein gels.

    Science.gov (United States)

    Banc, A; Charbonneau, C; Dahesh, M; Appavou, M-S; Fu, Z; Morel, M-H; Ramos, L

    2016-06-28

    We propose a quantitative approach to probe the spatial heterogeneities of interactions in macromolecular gels, based on a combination of small angle X-ray (SAXS) and neutrons (SANS) scattering. We investigate the structure of model gluten protein gels and show that the gels display radically different SAXS and SANS profiles when the solvent is (at least partially) deuterated. The detailed analysis of the SANS signal as a function of the solvent deuteration demonstrates heterogeneities of sample deuteration at different length scales. The progressive exchange between the protons (H) of the proteins and the deuteriums (D) of the solvent is inhomogeneous and 60 nm large zones that are enriched in H are evidenced. In addition, at low protein concentration, in the sol state, solvent deuteration induces a liquid/liquid phase separation. Complementary biochemical and structure analyses show that the denser protein phase is more protonated and specifically enriched in glutenin, the polymeric fraction of gluten proteins. These findings suggest that the presence of H-rich zones in gluten gels would arise from the preferential interaction of glutenin polymers through a tight network of non-exchangeable intermolecular hydrogen bonds. PMID:27198847

  6. Computational analysis of translational readthrough proteins in Drosophila and yeast reveals parallels to alternative splicing.

    Science.gov (United States)

    Pancsa, Rita; Macossay-Castillo, Mauricio; Kosol, Simone; Tompa, Peter

    2016-01-01

    In translational readthrough (TR) the ribosome continues extending the nascent protein beyond the first in-frame termination codon. Due to the lack of dedicated analyses of eukaryotic TR cases, the associated functional-evolutionary advantages are still unclear. Here, based on a variety of computational methods, we describe the structural and functional properties of previously proposed D. melanogaster and S. cerevisiae TR proteins and extensions. We found that in D. melanogaster TR affects long proteins in mainly regulatory roles. Their TR-extensions are structurally disordered and rich in binding motifs, which, together with their cell-type- and developmental stage-dependent inclusion, suggest that similarly to alternatively spliced exons they rewire cellular interaction networks in a temporally and spatially controlled manner. In contrast, yeast TR proteins are rather short and fulfil mainly housekeeping functions, like translation. Yeast extensions usually lack disorder and linear motifs, which precludes elucidating their functional relevance with sufficient confidence. Therefore we propose that by being much more restricted and by lacking clear functional hallmarks in yeast as opposed to fruit fly, TR shows remarkable parallels with alternative splicing. Additionally, the lack of conservation of TR extensions among orthologous TR proteins suggests that TR-mediated functions may be generally specific to lower taxonomic levels. PMID:27561673

  7. Proteomic Analysis of Saliva in HIV-positive Heroin Addicts Reveals Proteins Correlated with Cognition

    Energy Technology Data Exchange (ETDEWEB)

    Dominy, Stephen; Brown, Joseph N.; Ryder, Mark I.; Gritsenko, Marina A.; Jacobs, Jon M.; Smith, Richard D.

    2014-04-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last few years to account for this phenomenon, including the neurotoxic effects of antiretrovirals and co-morbid substance abuse. However, no underlying molecular mechanism has been identified. Emerging evidence in several fields has linked the gut to brain diseases, but the effect of the gut on the brain during HIV infection has not been explored. Saliva is the most accessible gut biofluid, and is therefore of great scientific interest for diagnostic and prognostic purposes. This study presents a longitudinal, liquid chromatography-mass spectrometry-based quantitative proteomics study investigating saliva samples taken from 8 HIV-positive (HIV+) and 11 -negative (HIV-) heroin addicts. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores and that implicate disruption of protein quality control pathways by HIV. Notably, no proteins from the HIV- heroin addict cohort showed significant correlations with cognitive scores. In addition, the majority of correlated proteins have been shown to be associated with exosomes, allowing us to propose that the salivary glands and/or oral epithelium may modulate brain function during HIV infection through the release of discrete packets of proteins in the form of exosomes.

  8. The presence of disulfide bonds reveals an evolutionarily conserved mechanism involved in mitochondrial protein translocase assembly

    Science.gov (United States)

    Wrobel, Lidia; Sokol, Anna M.; Chojnacka, Magdalena; Chacinska, Agnieszka

    2016-01-01

    Disulfide bond formation is crucial for the biogenesis and structure of many proteins that are localized in the intermembrane space of mitochondria. The importance of disulfide bond formation within mitochondrial proteins was extended beyond soluble intermembrane space proteins. Tim22, a membrane protein and core component of the mitochondrial translocase TIM22, forms an intramolecular disulfide bond in yeast. Tim22 belongs to the Tim17/Tim22/Tim23 family of protein translocases. Here, we present evidence of the high evolutionary conservation of disulfide bond formation in Tim17 and Tim22 among fungi and metazoa. Topological models are proposed that include the location of disulfide bonds relative to the predicted transmembrane regions. Yeast and human Tim22 variants that are not oxidized do not properly integrate into the membrane complex. Moreover, the lack of Tim17 oxidation disrupts the TIM23 translocase complex. This underlines the importance of disulfide bond formation for mature translocase assembly through membrane stabilization of weak transmembrane domains. PMID:27265872

  9. Crystal structure of the Campylobacter jejuni Cj0090 protein reveals a novel variant of the immunoglobulin fold among bacterial lipoproteins

    OpenAIRE

    Paek, Seonghee; Kawai, Fumihiro; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2012-01-01

    Bacterial lipoproteins play an important role in bacterial pathogenesis and physiology. The genome of Campylobacter jejuni, a major foodborn pathogen, is predicted to contain over 20 lipoproteins. However, the functions of the majority of C. jejuni lipoproteins remain unknown. The Cj0090 protein is encoded by a lipoprotein operon composed of cj0089, cj0090, and cj0091. Here, we report the crystal structure of Cj0090 at 1.9 Å resolution, revealing a novel variant of the immunoglobulin fold wit...

  10. Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins

    DEFF Research Database (Denmark)

    Cappellini, E.; Jensen, L.J.; Szklarczyk, D.; Ginolhac, A.; Da Fonseca, R.A.R.; Stafford, Thomas; Holen, S.R.; Collins, M.J.; Orlando, L.; Willerslev, E.; Gilbert, M Thomas P; Olsen, J.V.

    2012-01-01

    described beyond subpolar environments. Mass spectrometry-based ancient protein sequencing offers new perspectives for future molecular phylogenetic inference and physiological studies on samples not amenable to ancient DNA investigation. This approach therefore represents a further step into the ongoing......We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth (Mammuthus primigenius) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low...... evidence was observed of amino acid modifications due to post-mortem hydrolytic and oxidative damage. A consistent subset of this permafrost bone proteome was also identified in more recent Columbian mammoth (Mammuthus columbi) samples from temperate latitudes, extending the potential of the approach...

  11. Structural analysis of mitochondrial mutations reveals a role for bigenomic protein interactions in human disease.

    Directory of Open Access Journals (Sweden)

    Rhiannon E Lloyd

    Full Text Available Mitochondria are the energy producing organelles of the cell, and mutations within their genome can cause numerous and often severe human diseases. At the heart of every mitochondrion is a set of five large multi-protein machines collectively known as the mitochondrial respiratory chain (MRC. This cellular machinery is central to several processes important for maintaining homeostasis within cells, including the production of ATP. The MRC is unique due to the bigenomic origin of its interacting proteins, which are encoded in the nucleus and mitochondria. It is this, in combination with the sheer number of protein-protein interactions that occur both within and between the MRC complexes, which makes the prediction of function and pathological outcome from primary sequence mutation data extremely challenging. Here we demonstrate how 3D structural analysis can be employed to predict the functional importance of mutations in mtDNA protein-coding genes. We mined the MITOMAP database and, utilizing the latest structural data, classified mutation sites based on their location within the MRC complexes III and IV. Using this approach, four structural classes of mutation were identified, including one underexplored class that interferes with nuclear-mitochondrial protein interactions. We demonstrate that this class currently eludes existing predictive approaches that do not take into account the quaternary structural organization inherent within and between the MRC complexes. The systematic and detailed structural analysis of disease-associated mutations in the mitochondrial Complex III and IV genes significantly enhances the predictive power of existing approaches and our understanding of how such mutations contribute to various pathologies. Given the general lack of any successful therapeutic approaches for disorders of the MRC, these findings may inform the development of new diagnostic and prognostic biomarkers, as well as new drugs and targets for

  12. The structure of p53 tumour suppressor protein reveals the basis for its functional plasticity

    OpenAIRE

    Okorokov, A L; Sherman, M. B.; Plisson, C.; Grinkevich, V; Sigmundsson, K.; Selivanova, G; Milner, J.; E V Orlova

    2006-01-01

    p53 major tumour suppressor protein has presented a challenge for structural biology for two decades. The intact and complete p53 molecule has eluded previous attempts to obtain its structure, largely due to the intrinsic flexibility of the protein. Using ATP-stabilised p53, we have employed cryoelectron microscopy and single particle analysis to solve the first three-dimensional structure of the full-length p53 tetramer (resolution 13.7 A). The p53 molecule is a D2 tetramer, resembling a hol...

  13. Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Khotin, Mikhail, E-mail: h_mg@mail.ru [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Turoverova, Lidia [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Aksenova, Vasilisa [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Department of Genetics, St. Petersburg State University, Universitetskaya nab., 7/9, 199034 St. Petersburg (Russian Federation); Barlev, Nikolai [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN (United Kingdom); Borutinskaite, Veronika Viktorija [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Department of Developmental Biology, Institute of Biochemistry, LT-08662 Vilnius (Lithuania); Vener, Alexander [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Bajenova, Olga [Department of Genetics, St. Petersburg State University, Universitetskaya nab., 7/9, 199034 St. Petersburg (Russian Federation); Magnusson, Karl-Eric [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Pinaev, George P. [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Tentler, Dmitri, E-mail: dtentler@mail.cytspb.rssi.ru [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation)

    2010-06-25

    Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

  14. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David S.

    2014-07-09

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  15. Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle BiogenesisW⃞

    Science.gov (United States)

    Lurin, Claire; Andrés, Charles; Aubourg, Sébastien; Bellaoui, Mohammed; Bitton, Frédérique; Bruyère, Clémence; Caboche, Michel; Debast, Cédrig; Gualberto, José; Hoffmann, Beate; Lecharny, Alain; Le Ret, Monique; Martin-Magniette, Marie-Laure; Mireau, Hakim; Peeters, Nemo; Renou, Jean-Pierre; Szurek, Boris; Taconnat, Ludivine; Small, Ian

    2004-01-01

    The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms. PMID:15269332

  16. Revealing the role of oxidation state in interaction between nitro/amino-derived particulate matter and blood proteins

    Science.gov (United States)

    Liu, Zhen; Li, Ping; Bian, Weiwei; Yu, Jingkai; Zhan, Jinhua

    2016-05-01

    Surface oxidation states of ultrafine particulate matter can influence the proinflammatory responses and reactive oxygen species levels in tissue. Surface active species of vehicle-emission soot can serve as electron transfer-mediators in mitochondrion. Revealing the role of surface oxidation state in particles-proteins interaction will promote the understanding on metabolism and toxicity. Here, the surface oxidation state was modeled by nitro/amino ligands on nanoparticles, the interaction with blood proteins were evaluated by capillary electrophoresis quantitatively. The nitro shown larger affinity than amino. On the other hand, the affinity to hemoglobin is 103 times larger than that to BSA. Further, molecular docking indicated the difference of binding intensity were mainly determined by hydrophobic forces and hydrogen bonds. These will deepen the quantitative understanding of protein-nanoparticles interaction from the perspective of surface chemical state.

  17. Salivary gland proteome analysis reveals modulation of anopheline unique proteins in insensitive acetylcholinesterase resistant Anopheles gambiae mosquitoes.

    Directory of Open Access Journals (Sweden)

    Sylvie Cornelie

    Full Text Available Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1(R allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1(R resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa.

  18. Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Haijun [Washington University; Zhang, Hao [Washington University; King, Jeremy D. [Washington University; Wolf, Nathan R. [Washington University; Prado, Mindy [Washington University; Gross, Michael L. [Washington University; Blankenship, Robert E. [Washington University

    2014-12-01

    The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outside of the β-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.

  19. Phosphoproteome analysis of streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation

    DEFF Research Database (Denmark)

    Manteca, Angel; Ye, Juanying; Sánchez, Jesús;

    2011-01-01

    bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation...

  20. Involvement of surfactant protein D in emphysema revealed by genetic association study

    OpenAIRE

    Ishii, Takeo; Hagiwara, Koichi; Kamio, Koichiro; Ikeda, Shinobu; Arai, Tomio; Mieno, Makiko Naka; Kumasaka, Toshio; Muramatsu, Masaaki; Sawabe, Motoji; Gemma, Akihiko; Kida, Kozui

    2011-01-01

    Surfactant protein D (SFTPD) induces emphysema in knockout mice, but the association of SFTPD with chronic obstructive pulmonary disease (COPD) and emphysema in humans is unclear. Therefore, we aimed to determine the association between genetic variations in SFTPD and susceptibility to COPD and emphysema.

  1. Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation

    Czech Academy of Sciences Publication Activity Database

    Tylečková, Jiřina; Valeková, Ivona; Žižková, Martina; Rákocyová, Michaela; Maršala, S.; Maršala, M.; Gadher, S. J.; Kovářová, Hana

    2016-01-01

    Roč. 132, č. 1 (2016), s. 13-20. ISSN 1874-3919 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : cell adhesion proteins * cell surface capture * neuronal differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.888, year: 2014

  2. Functional genomics of tick thioester-containing proteins reveal the ancient origin of the complement system

    Czech Academy of Sciences Publication Activity Database

    Burešová, Veronika; Hajdušek, Ondřej; Franta, Zdeněk; Loosová, Gabriela; Grunclová, Lenka; Levashina, E.A.; Kopáček, Petr

    2011-01-01

    Roč. 3, č. 6 (2011), s. 623-630. ISSN 1662-811X R&D Projects: GA ČR GAP506/10/2136; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z60220518 Keywords : tick * thioester-containing proteins * complement Subject RIV: EC - Immunology Impact factor: 4.209, year: 2011

  3. Local and non-local native topologies reveal the underlying folding landscape of proteins

    International Nuclear Information System (INIS)

    Due to Plaxco, Simons, Baker and others, it is now well known that the two-state single domain protein folding rate is fairly well predicted from knowledge of the topology of the native structure. Plaxco et al found that the folding rates of two-state proteins correlate with the average degree to which native contacts are 'local' within the chain sequence: fast-folders usually have mostly local structures. Here, we dissected the native topology further by focusing on non-local and local contacts using lower and upper bounds of allowable sequence separation in computing the average contact order. We analyzed non-local and local contacts of 82 two-state proteins whose experimental folding rates span over six orders of magnitude. We observed that both the number of non-local contacts and the average sequence separation of non-local contacts (non-local CO) are both negatively correlated with the folding rate, showing that the non-local contacts dominate the barrier-crossing process. Surprisingly, the local contact orders of the proteins also correlate with the folding rates. However, this correlation shows a strong positive trend indicating the role of a diffusive search in the denatured basin

  4. Affinity chromatography reveals RuBisCO as an ecdysteroid-binding protein

    Czech Academy of Sciences Publication Activity Database

    Uhlík, Ondřej; Kamlar, Marek; Kohout, Ladislav; Ježek, Rudolf; Harmatha, Juraj; Macek, Tomáš

    2008-01-01

    Roč. 73, č. 14 (2008), s. 1433-1440. ISSN 0039-128X R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z40550506 Keywords : ecdysteroids * RuBisCO * regulation * binding protein Subject RIV: CC - Organic Chemistry Impact factor: 2.588, year: 2008

  5. Chemical genetics reveals an RGS/G-protein role in the action of a compound.

    Directory of Open Access Journals (Sweden)

    Kevin Fitzgerald

    2006-04-01

    Full Text Available We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR signaling involving G-alphaq (G-protein alpha subunit. Our studies suggest that the small molecules act at the level of the RGS/G-alphaq signaling complex, and define new mutations in both RGS and G-alphaq, including a unique hypo-adapation allele of G-alphaq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.

  6. Distinct configurations of protein complexes and biochemical pathways revealed by epistatic interaction network motifs

    LENUS (Irish Health Repository)

    Casey, Fergal

    2011-08-22

    Abstract Background Gene and protein interactions are commonly represented as networks, with the genes or proteins comprising the nodes and the relationship between them as edges. Motifs, or small local configurations of edges and nodes that arise repeatedly, can be used to simplify the interpretation of networks. Results We examined triplet motifs in a network of quantitative epistatic genetic relationships, and found a non-random distribution of particular motif classes. Individual motif classes were found to be associated with different functional properties, suggestive of an underlying biological significance. These associations were apparent not only for motif classes, but for individual positions within the motifs. As expected, NNN (all negative) motifs were strongly associated with previously reported genetic (i.e. synthetic lethal) interactions, while PPP (all positive) motifs were associated with protein complexes. The two other motif classes (NNP: a positive interaction spanned by two negative interactions, and NPP: a negative spanned by two positives) showed very distinct functional associations, with physical interactions dominating for the former but alternative enrichments, typical of biochemical pathways, dominating for the latter. Conclusion We present a model showing how NNP motifs can be used to recognize supportive relationships between protein complexes, while NPP motifs often identify opposing or regulatory behaviour between a gene and an associated pathway. The ability to use motifs to point toward underlying biological organizational themes is likely to be increasingly important as more extensive epistasis mapping projects in higher organisms begin.

  7. The spliceosomal snRNP core complex of Trypanosoma brucei: Cloning and functional analysis reveals seven Sm protein constituents

    Science.gov (United States)

    Palfi, Zsofia; Lücke, Stephan; Lahm, Hans-Werner; Lane, William S.; Kruft, Volker; Bragado-Nilsson, Elisabeth; Séraphin, Bertrand; Bindereif, Albrecht

    2000-01-01

    Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4/U6, and U5, as well as the spliced leader (SL) RNP, contains a core of common proteins, which we have previously identified. This core is unusual because it is not recognized by anti-Sm Abs and it associates with an Sm-related sequence in the trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common proteins of 8.5, 10, 12.5, 14, and 15 kDa of Trypanosoma brucei and identified them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and -D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T. brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus completing a set of seven canonical Sm proteins. Sequence comparisons of the trypanosome proteins revealed several deviations in highly conserved positions from the Sm consensus motif. We have identified a network of specific heterodimeric and -trimeric Sm protein interactions in vitro. These results are summarized in a model of the trypanosome Sm core, which argues for a strong conservation of the Sm particle structure. The conservation extends also to the functional level, because at least one trypanosome Sm protein, SmG, was able to specifically complement a corresponding mutation in yeast. PMID:10900267

  8. Comparative analysis reveals conserved protein phosphorylation networks implicated in multiple diseases

    DEFF Research Database (Denmark)

    Tan, Chris Soon Heng; Bodenmiller, Bernd; Pasculescu, Adrian;

    2009-01-01

    Protein kinases enable cellular information processing. Although numerous human phosphorylation sites and their dynamics have been characterized, the evolutionary history and physiological importance of many signaling events remain unknown. Using target phosphoproteomes determined with a similar...... experimental and computational pipeline, we investigated the conservation of human phosphorylation events in distantly related model organisms (fly, worm, and yeast). With a sequence-alignment approach, we identified 479 phosphorylation events in 344 human proteins that appear to be positionally conserved over...... approximately 600 million years of evolution and hence are likely to be involved in fundamental cellular processes. This sequence-alignment analysis suggested that many phosphorylation sites evolve rapidly and therefore do not display strong evolutionary conservation in terms of sequence position in distantly...

  9. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

    Energy Technology Data Exchange (ETDEWEB)

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

    2010-09-21

    Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

  10. Nuclear magnetic resonance-based metabolomics reveals that dairy protein fractions affect urinary urea excretion differently in overweight adolescents

    DEFF Research Database (Denmark)

    Zheng, Hong; Yde, Christian Clement; Dalsgaard, Trine Kastrup;

    2015-01-01

    Dairy proteins are an important part of our diet, and recently, there is considerable focus on understanding the effects of the two major dairy proteins fractions constituted by casein and whey. In the present study, the impact of a dietary intervention with casein, whey, and skim milk was invest...... to disclosure exposure effects. In conclusion, the present study demonstrates that casein and whey proteins impact urinary urea excretion differently.......Dairy proteins are an important part of our diet, and recently, there is considerable focus on understanding the effects of the two major dairy proteins fractions constituted by casein and whey. In the present study, the impact of a dietary intervention with casein, whey, and skim milk was...... excretion of urea was found after the 12-week casein and skim milk interventions, while the 12-week whey intervention had no significant effect on the urea excretion. In addition, NMR-based metabolomics revealed a decreased urinary citrate excretion in the whey group and thereby demonstrated its potential...

  11. Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation

    Czech Academy of Sciences Publication Activity Database

    Tylečková, Jiřina; Valeková, Ivona; Žižková, Martina; Rákocyová, Michaela; Maršala, S.; Maršala, M.; Gadher, S. J.; Kovářová, Hana

    Poznaň : PTP, 2015. s. 1-1. [Central and Eastern European Proteomic Conference (CEEPC) /9./. 15.06.2015-18.06.2015, Poznaň] R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : cell adhesion proteins * cell surface capture * neuronal differentiation Subject RIV: EB - Genetics ; Molecular Biology

  12. Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development

    OpenAIRE

    Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Bjorn; Moritz, Thomas; Hvidsten, Torgeir R.; Trygg, Johan; Wingsle, Gunnar

    2016-01-01

    Background: Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and var...

  13. Accelerated protein evolution analysis reveals genes and pathways associated with the evolution of mammalian longevity

    OpenAIRE

    Li, Yang; de Magalhães, João Pedro

    2011-01-01

    The genetic basis of the large species differences in longevity and aging remains a mystery. Thanks to recent large-scale genome sequencing efforts, the genomes of multiple species have been sequenced and can be used for cross-species comparisons to study species divergence in longevity. By analyzing proteins under accelerated evolution in several mammalian lineages where maximum lifespan increased, we identified genes and processes that are candidate targets of selection when longevity evolv...

  14. Novel serum protein biomarker panel revealed by mass spectrometry and its prognostic value in breast cancer

    OpenAIRE

    Chung, Liping; Moore, Katrina; Phillips, Leo; Boyle, Frances M.; Marsh, Deborah J.; Baxter, Robert C.

    2014-01-01

    Introduction Serum profiling using proteomic techniques has great potential to detect biomarkers that might improve diagnosis and predict outcome for breast cancer patients (BC). This study used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) to identify differentially expressed proteins in sera from BC and healthy volunteers (HV), with the goal of developing a new prognostic biomarker panel. Methods Training set serum samples from 99 BC and 51 H...

  15. The Population Genomics of Sunflowers and Genomic Determinants of Protein Evolution Revealed by RNAseq

    OpenAIRE

    Rieseberg, Loren H.; Kane, Nolan C.; Brook T. Moyers; Sébastien Renaut; Grassa, Christopher J.

    2012-01-01

    Few studies have investigated the causes of evolutionary rate variation among plant nuclear genes, especially in recently diverged species still capable of hybridizing in the wild. The recent advent of Next Generation Sequencing (NGS) permits investigation of genome wide rates of protein evolution and the role of selection in generating and maintaining divergence. Here, we use individual whole-transcriptome sequencing (RNAseq) to refine our understanding of the population genomics of wild spe...

  16. Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria

    OpenAIRE

    Gefen, Orit; Gabay, Chana; Mumcuoglu, Michael; Engel, Giora; Balaban, Nathalie Q.

    2008-01-01

    Phenotypic variability in populations of cells has been linked to evolutionary robustness to stressful conditions. A remarkable example of the importance of cell-to-cell variability is found in bacterial persistence, where subpopulations of dormant bacteria, termed persisters, were shown to be responsible for the persistence of the population to antibiotic treatments. Here, we use microfluidic devices to monitor the induction of fluorescent proteins under synthetic promoters and characterize ...

  17. Protein profiling reveals consequences of lifestyle choices on predicted biological aging

    OpenAIRE

    Stefan Enroth; Sofia Bosdotter Enroth; Åsa Johansson; Ulf Gyllensten

    2015-01-01

    Ageing is linked to a number of changes in how the body and its organs function. On a molecular level, ageing is associated with a reduction of telomere length, changes in metabolic and gene-transcription profiles and an altered DNA-methylation pattern. Lifestyle factors such as smoking or stress can impact some of these molecular processes and thereby affect the ageing of an individual. Here we demonstrate by analysis of 77 plasma proteins in 976 individuals, that the abundance of circulatin...

  18. Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization.

    Directory of Open Access Journals (Sweden)

    Valeriy Demchev

    Full Text Available Fibrinogen like protein 1(Fgl1 is a secreted protein with mitogenic activity on primary hepatocytes. Fgl1 is expressed in the liver and its expression is enhanced following acute liver injury. In animals with acute liver failure, administration of recombinant Fgl1 results in decreased mortality supporting the notion that Fgl1 stimulates hepatocyte proliferation and/or protects hepatocytes from injury. However, because Fgl1 is secreted and detected in the plasma, it is possible that the role of Fgl1 extends far beyond its effect on hepatocytes. In this study, we show that Fgl1 is additionally expressed in brown adipose tissue. We find that signals elaborated following liver injury also enhance the expression of Fgl1 in brown adipose tissue suggesting that there is a cross talk between the injured liver and adipose tissues. To identify extra hepatic effects, we generated Fgl1 deficient mice. These mice exhibit a phenotype suggestive of a global metabolic defect: Fgl1 null mice are heavier than wild type mates, have abnormal plasma lipid profiles, fasting hyperglycemia with enhanced gluconeogenesis and exhibit differences in white and brown adipose tissue morphology when compared to wild types. Because Fgl1 shares structural similarity to Angiopoietin like factors 2, 3, 4 and 6 which regulate lipid metabolism and energy utilization, we postulate that Fgl1 is a member of an emerging group of proteins with key roles in metabolism and liver regeneration.

  19. Protein profiling reveals consequences of lifestyle choices on predicted biological aging.

    Science.gov (United States)

    Enroth, Stefan; Enroth, Sofia Bosdotter; Johansson, Åsa; Gyllensten, Ulf

    2015-01-01

    Ageing is linked to a number of changes in how the body and its organs function. On a molecular level, ageing is associated with a reduction of telomere length, changes in metabolic and gene-transcription profiles and an altered DNA-methylation pattern. Lifestyle factors such as smoking or stress can impact some of these molecular processes and thereby affect the ageing of an individual. Here we demonstrate by analysis of 77 plasma proteins in 976 individuals, that the abundance of circulating proteins accurately predicts chronological age, as well as anthropometrical measurements such as weight, height and hip circumference. The plasma protein profile can also be used to identify lifestyle factors that accelerate and decelerate ageing. We found smoking, high BMI and consumption of sugar-sweetened beverages to increase the predicted chronological age by 2-6 years, while consumption of fatty fish, drinking moderate amounts of coffee and exercising reduced the predicted age by approximately the same amount. This method can be applied to dried blood spots and may thus be useful in forensic medicine to provide basic anthropometrical measures for an individual based on a biological evidence sample. PMID:26619799

  20. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    International Nuclear Information System (INIS)

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-β-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption

  1. Comparative proteomic analysis reveals mite (Varroa destructor) resistance-related proteins in Eastern honeybees (Apis cerana).

    Science.gov (United States)

    Ji, T; Shen, F; Liu, Z; Yin, L; Shen, J; Liang, Q; Luo, Y X

    2015-01-01

    The mite (Varroa destructor) has become the greatest threat to apiculture worldwide. As the original host of the mite, Apis cerana can effectively resist the mite. An increased understanding of the resistance mechanisms of Eastern honeybees against V. destructor may help researchers to protect other species against these parasites. In this study, the proteomes of 4 Apis cerana colonies were analyzed using an isobaric tag for relative and absolute quantitation technology. We determined the differences in gene and protein expression between susceptible and resistant colonies that were either unchallenged or challenged by V. destructor. The results showed that a total of 1532 proteins were identified. Gene Ontology enrichment analysis suggested that the transcription factors and basic metabolic and respiratory processes were efficient and feasible factors controlling this resistance, and 12 differentially expressed proteins were identified in Venn analysis. The results were validated by quantitative polymerase chain reaction. This study may provide insight into the genetic mechanisms underlying the resistance of honeybee to mites. PMID:26345948

  2. Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria.

    Science.gov (United States)

    Gefen, Orit; Gabay, Chana; Mumcuoglu, Michael; Engel, Giora; Balaban, Nathalie Q

    2008-04-22

    Phenotypic variability in populations of cells has been linked to evolutionary robustness to stressful conditions. A remarkable example of the importance of cell-to-cell variability is found in bacterial persistence, where subpopulations of dormant bacteria, termed persisters, were shown to be responsible for the persistence of the population to antibiotic treatments. Here, we use microfluidic devices to monitor the induction of fluorescent proteins under synthetic promoters and characterize the dormant state of single persister bacteria. Surprisingly, we observe that protein production does take place in supposedly dormant bacteria, over a narrow time window after the exit from stationary phase. Only thereafter does protein production stop, suggesting that differentiation into persisters fully develops over this time window and not during starvation, as previously believed. In effect, we observe that exposure of bacteria to antibiotics during this time window significantly reduces persistence. Our results point to new strategies to fight persistent bacterial infections. The quantitative measurement of single-cell induction presented in this study should shed light on the processes leading to the dormancy of subpopulations in different systems, such as in subpopulations of viable but nonculturable bacteria, or those of quiescent cancer cells. PMID:18427112

  3. Proteomic Analysis Reveals Key Proteins and Phosphoproteins upon Seed Germination of Wheat (Triticum aestivum L.).

    Science.gov (United States)

    Dong, Kun; Zhen, Shoumin; Cheng, Zhiwei; Cao, Hui; Ge, Pei; Yan, Yueming

    2015-01-01

    Wheat (Triticum aestivum L.) is one of the oldest cultivated crops and the second most important food crop in the world. Seed germination is the key developmental process in plant growth and development, and poor germination directly affects plant growth and subsequent grain yield. In this study, we performed the first dynamic proteome analysis of wheat seed germination using a two-dimensional differential gel electrophoresis (2D-DIGE)-based proteomic approach. A total of 166 differentially expressed protein (DEP) spots representing 73 unique proteins were identified, which are mainly involved in storage, stress/defense/detoxification, carbohydrate metabolism, photosynthesis, cell metabolism, and transcription/translation/transposition. The identified DEPs and their dynamic expression profiles generally correspond to three distinct seed germination phases after imbibition: storage degradation, physiological processes/morphogenesis, and photosynthesis. Some key DEPs involved in storage substance degradation and plant defense mechanisms, such as globulin 3, sucrose synthase type I, serpin, beta-amylase, and plastid ADP-glucose pyrophosphorylase (AGPase) small subunit, were found to be phosphorylated during seed germination. Particularly, the phosphorylation site Ser(355) was found to be located in the enzyme active region of beta-amylase, which promotes substrate binding. Phosphorylated modification of several proteins could promote storage substance degradation and environmental stress defense during seed germination. The central metabolic pathways involved in wheat seed germination are proposed herein, providing new insights into the molecular mechanisms of cereal seed germination. PMID:26635843

  4. Isolation, characterization, and bioinformatic analysis of calmodulin-binding protein cmbB reveals a novel tandem IP22 repeat common to many Dictyostelium and Mimivirus proteins.

    Science.gov (United States)

    O'Day, Danton H; Suhre, Karsten; Myre, Michael A; Chatterjee-Chakraborty, Munmun; Chavez, Sara E

    2006-08-01

    A novel calmodulin-binding protein cmbB from Dictyostelium discoideum is encoded in a single gene. Northern analysis reveals two cmbB transcripts first detectable at 4 h during multicellular development. Western blotting detects an approximately 46.6 kDa protein. Sequence analysis and calmodulin-agarose binding studies identified a "classic" calcium-dependent calmodulin-binding domain (179IPKSLRSLFLGKGYNQPLEF198) but structural analyses suggest binding may not involve classic alpha-helical calmodulin-binding. The cmbB protein is comprised of tandem repeats of a newly identified IP22 motif ([I,L]Pxxhxxhxhxxxhxxxhxxxx; where h = any hydrophobic amino acid) that is highly conserved and a more precise representation of the FNIP repeat. At least eight Acanthamoeba polyphaga Mimivirus proteins and over 100 Dictyostelium proteins contain tandem arrays of the IP22 motif and its variants. cmbB also shares structural homology to YopM, from the plague bacterium Yersenia pestis. PMID:16777069

  5. Functional protein network activation mapping reveals new potential molecular drug targets for poor prognosis pediatric BCP-ALL.

    Directory of Open Access Journals (Sweden)

    Benedetta Accordi

    Full Text Available BACKGROUND: In spite of leukemia therapy improvements obtained over the last decades, therapy is not yet effective in all cases. Current approaches in Acute Lymphoblastic Leukemia (ALL research focus on identifying new molecular targets to improve outcome for patients with a dismal prognosis. In this light phosphoproteomics seems to hold great promise for the identification of proteins suitable for targeted therapy. METHODOLOGY/PRINCIPAL FINDINGS: We employed Reverse Phase Protein Microarrays to identify aberrantly activated proteins in 118 pediatric B-cell precursor (BCP-ALL patients. Signal transduction pathways were assayed for activation/expression status of 92 key signalling proteins. We observed an increased activation/expression of several pathways involved in cell proliferation in poor clinical prognosis patients. MLL-rearranged tumours revealed BCL-2 hyperphosphorylation through AMPK activation, which indicates that AMPK could provide a functional role in inhibiting apoptosis in MLL-rearranged patients, and could be considered as a new potential therapeutic target. Second, in patients with poor clinical response to prednisone we observed the up-modulation of LCK activity with respect to patients with good response. This tyrosine-kinase can be down-modulated with clinically used inhibitors, thus modulating LCK activity could be considered for further studies as a new additional therapy for prednisone-resistant patients. Further we also found an association between high levels of CYCLIN E and relapse incidence. Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis. CONCLUSIONS/SIGNIFICANCE: We conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.

  6. Protein Interaction Networks Reveal Novel Autism Risk Genes within GWAS Statistical Noise

    Science.gov (United States)

    Correia, Catarina; Oliveira, Guiomar; Vicente, Astrid M.

    2014-01-01

    Genome-wide association studies (GWAS) for Autism Spectrum Disorder (ASD) thus far met limited success in the identification of common risk variants, consistent with the notion that variants with small individual effects cannot be detected individually in single SNP analysis. To further capture disease risk gene information from ASD association studies, we applied a network-based strategy to the Autism Genome Project (AGP) and the Autism Genetics Resource Exchange GWAS datasets, combining family-based association data with Human Protein-Protein interaction (PPI) data. Our analysis showed that autism-associated proteins at higher than conventional levels of significance (P<0.1) directly interact more than random expectation and are involved in a limited number of interconnected biological processes, indicating that they are functionally related. The functionally coherent networks generated by this approach contain ASD-relevant disease biology, as demonstrated by an improved positive predictive value and sensitivity in retrieving known ASD candidate genes relative to the top associated genes from either GWAS, as well as a higher gene overlap between the two ASD datasets. Analysis of the intersection between the networks obtained from the two ASD GWAS and six unrelated disease datasets identified fourteen genes exclusively present in the ASD networks. These are mostly novel genes involved in abnormal nervous system phenotypes in animal models, and in fundamental biological processes previously implicated in ASD, such as axon guidance, cell adhesion or cytoskeleton organization. Overall, our results highlighted novel susceptibility genes previously hidden within GWAS statistical “noise” that warrant further analysis for causal variants. PMID:25409314

  7. Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2

    Science.gov (United States)

    Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

    2016-03-01

    Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research.

  8. Survey of large protein complexes D. vulgaris reveals great structural diversity

    Energy Technology Data Exchange (ETDEWEB)

    Han, B.-G.; Dong, M.; Liu, H.; Camp, L.; Geller, J.; Singer, M.; Hazen, T. C.; Choi, M.; Witkowska, H. E.; Ball, D. A.; Typke, D.; Downing, K. H.; Shatsky, M.; Brenner, S. E.; Chandonia, J.-M.; Biggin, M. D.; Glaeser, R. M.

    2009-08-15

    An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr {approx} 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate {approx}10 times greater than that of previous 'proteomic' screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.

  9. Protein dynamics revealed in the excitonic spectra of single LH2 complexes

    International Nuclear Information System (INIS)

    The fluorescence emission spectrum of single peripheral light-harvesting (LH2) complexes of the photosynthetic purple bacterium Rhodopseudomonas acidophila exhibits remarkable dynamics on a time scale of several minutes. Often the spectral properties are quasi-stable; sometimes large spectral jumps to the blue or to the red are observed. To explain the dynamics, every pigment is proposed to be in two conformational substates with different excitation energies, which originate from the conformational state of the protein as a result of pigment-protein interaction. Due to the excitonic coupling in the ring of 18 pigments, the two-state assumption generates a substantial amount of distinct spectroscopic states, which reflect part of the inhomogeneous distributed spectral properties of LH2. To describe the observed dynamics, spontaneous and light-induced transitions are introduced between the two states. For each 'realization of the disorder', the spectral properties are calculated using a disordered exciton model combined with the modified Redfield theory to obtain realistic spectral line shapes. The single-molecule fluorescence peak (FLP) distribution, the distribution dependence on the excitation intensity, and the FLP time traces are well described within the framework of this model

  10. Proteins with altered levels in plasma from glioblastoma patients as revealed by iTRAQ-based quantitative proteomic analysis.

    Directory of Open Access Journals (Sweden)

    Poonam Gautam

    Full Text Available Glioblastomas (GBMs are the most common and lethal primary tumors of the central nervous system with high level of recurrence despite aggressive therapy. Tumor-associated proteins/peptides may appear in the plasma of these patients as a result of disruption of the blood-brain barrier in them, raising the scope for development of plasma-based tests for diagnosis and monitoring the disease. With this objective, we analyzed the levels of proteins present in the plasma from GBM patients using an iTRAQ based LC-MS/MS approach. Analysis with pooled plasma specimens from the patient and healthy control samples revealed high confidence identification of 296 proteins, of which 61 exhibited a fold-change ≥1.5 in the patient group. Forty-eight of them contained signal sequence. A majority have been reported in the differentially expressed transcript or protein profile of GBM tissues; 6 have been previously studied as plasma biomarkers for GBM and 16 for other types of cancers. Altered levels of three representative proteins-ferritin light chain (FTL, S100A9, and carnosinase 1 (CNDP1-were verified by ELISA in a test set of ten individual plasma specimens. FTL is an inflammation marker also implicated in cancer, S100A9 is an important member of the Ca(2+ signaling cascade reported to be altered in GBM tissue, and CNDP1 has been reported for its role in the regulation of the levels of carnosine, implicated as a potential drug for GBM. These and other proteins in the dataset may form useful starting points for further clinical investigations for the development of plasma-based biomarker panels for GBM.

  11. Transcriptomic and Protein Expression Analysis Reveals Clinicopathological Significance of Bloom Syndrome Helicase (BLM) in Breast Cancer.

    Science.gov (United States)

    Arora, Arvind; Abdel-Fatah, Tarek M A; Agarwal, Devika; Doherty, Rachel; Moseley, Paul M; Aleskandarany, Mohammed A; Green, Andrew R; Ball, Graham; Alshareeda, Alaa T; Rakha, Emad A; Chan, Stephen Y T; Ellis, Ian O; Madhusudan, Srinivasan

    2015-04-01

    Bloom syndrome helicase (BLM) has key roles in homologous recombination repair, telomere maintenance, and DNA replication. Germ-line mutations in the BLM gene causes Bloom syndrome, a rare disorder characterized by premature aging and predisposition to multiple cancers, including breast cancer. The clinicopathologic significance of BLM in sporadic breast cancers is unknown. We investigated BLM mRNA expression in the Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1,950) and validated in an external dataset of 2,413 tumors. BLM protein level was evaluated in the Nottingham Tenovus series comprising 1,650 breast tumors. BLM mRNA overexpression was significantly associated with high histologic grade, larger tumor size, estrogen receptor-negative (ER(-)), progesterone receptor-negative (PR(-)), and triple-negative phenotypes (ps < 0.0001). BLM mRNA overexpression was also linked to aggressive molecular phenotypes, including PAM50.Her2 (P < 0.0001), PAM50.Basal (P < 0.0001), and PAM50.LumB (P < 0.0001) and Genufu subtype (ER(+)/Her2(-)/high proliferation; P < 0.0001). PAM50.LumA tumors and Genufu subtype (ER(+)/Her2(-)/low proliferation) were more likely to express low levels of BLM mRNA (ps < 0.0001). Integrative molecular clusters (intClust) intClust.1 (P < 0.0001), intClust.5 (P < 0.0001), intClust.9 (P < 0.0001), and intClust.10 (P < 0.0001) were also more likely in tumors with high BLM mRNA expression. BLM mRNA overexpression was associated with poor breast cancer-specific survival (BCSS; ps < 0.000001). At the protein level, altered subcellular localization with high cytoplasmic BLM and low nuclear BLM was linked to aggressive phenotypes. In multivariate analysis, BLM mRNA and BLM protein levels independently influenced BCSS. This is the first and the largest study to provide evidence that BLM is a promising biomarker in breast cancer. PMID:25673821

  12. An estrogen-responsive plasma protein expression signature in Atlantic cod (Gadus morhua) revealed by SELDI-TOF MS

    DEFF Research Database (Denmark)

    Nielsen, Mari Mæland; Meyer, Sonnich; Larsen, Bodil Katrine; Andersen, Odd Ketil; Hjelle, Anne

    2011-01-01

    Compound-specific protein expression signatures( PESs) can be revealed by proteomic techniques. The SELDI-TOF MS approach is advantageous due to its simplicity and high-throughput capacity,however, there are concerns regarding the reproducibility of this method. The aim of this study was to define...... an estrogen-responsive PES in plasma of Atlantic cod (Gadus morhua) using the SELDI-TOF MS technique. Protein expression analysis of male cod exposed to 17 b-estradiol (E2) showed that 27 plasma peaks were differentially expressed following exposure.There producibility of this result was evaluated by...... targeted antibody-assisted SELDI-TOF MS approach was carried out in an attempt to identify the E2-responsive peaks. Results indicated that 2 peaks were fragments of the well-known biomarkers VTG and/or ZRP. In this study, the SELDI-TOF MS technology has shown its potential for defining compound...

  13. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions.

    Science.gov (United States)

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G; Módos, Károly; Kardos, József; Liliom, Károly

    2010-10-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin-sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well. PMID:20522785

  14. The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

    Energy Technology Data Exchange (ETDEWEB)

    Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.; Garboczi, David N. (NIH)

    2010-11-03

    The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

  15. Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles

    Science.gov (United States)

    Wissenbach, Dirk K.; Pfeiffer, Susanne E. M.; Baumann, Sven; Hofbauer, Lorenz C.; von Bergen, Martin; Kalkhof, Stefan; Rammelt, Stefan

    2016-01-01

    Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis. PMID:27441377

  16. Structures of Adnectin/Protein Complexes Reveal an Expanded Binding Footprint

    Energy Technology Data Exchange (ETDEWEB)

    Ramamurthy, Vidhyashankar; Krystek, Jr., Stanley R.; Bush, Alexander; Wei, Anzhi; Emanuel, Stuart L.; Gupta, Ruchira Das; Janjua, Ahsen; Cheng, Lin; Murdock, Melissa; Abramczyk, Bozena; Cohen, Daniel; Lin, Zheng; Morin, Paul; Davis, Jonathan H.; Dabritz, Michael; McLaughlin, Douglas C.; Russo, Katie A.; Chao, Ginger; Wright, Martin C.; Jenny, Victoria A.; Engle, Linda J.; Furfine, Eric; Sheriff, Steven (BMS)

    2014-10-02

    Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin ({sup 10}Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three {sup 10}Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the {beta} strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these {beta} strand interactions, indicating that these nonloop residues can expand the available binding footprint.

  17. Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life

    DEFF Research Database (Denmark)

    Lynnerup, Niels; Kjeldsen, Henrik; Heegaard, Steffen;

    2008-01-01

    subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its (14)C content from the atmospheric carbon dioxide. The (14)C content of the lens proteins thus reflects the atmospheric content of (14)C when the lens crystallines were formed. Precise radiocarbon...... dating is made possible by comparing the (14)C content of the lens crystallines to the so-called bomb pulse, i.e. a plot of the atmospheric (14)C content since the Second World War, when there was a significant increase due to nuclear-bomb testing. Since the change in concentration is significant even on...... a yearly basis this allows very accurate dating. METHODOLOGY/PRINCIPAL FINDINGS: Our results allow us to conclude that the crystalline formation in the lens nucleus almost entirely takes place around the time of birth, with a very small, and decreasing, continuous formation throughout life. The...

  18. Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhou [ORNL; Czarnecki, Olaf [ORNL; Chourey, Karuna [ORNL; Yang, Jun [ORNL; Tuskan, Gerald A [ORNL; Hurst, Gregory {Greg} B [ORNL; Pan, Chongle [ORNL; Chen, Jay [ORNL

    2014-01-01

    Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. Here, a quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found SLs regulate the expression of about three dozens of proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

  19. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    International Nuclear Information System (INIS)

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH2-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

  20. A targeted library screen reveals a new inhibitor scaffold for protein kinase D.

    Directory of Open Access Journals (Sweden)

    Manuj Tandon

    Full Text Available Protein kinase D (PKD has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.

  1. Mimivirus reveals Mre11/Rad50 fusion proteins with a sporadic distribution in eukaryotes, bacteria, viruses and plasmids

    Directory of Open Access Journals (Sweden)

    Ogata Hiroyuki

    2011-09-01

    Full Text Available Abstract Background The Mre11/Rad50 complex and the homologous SbcD/SbcC complex in bacteria play crucial roles in the metabolism of DNA double-strand breaks, including DNA repair, genome replication, homologous recombination and non-homologous end-joining in cellular life forms and viruses. Here we investigated the amino acid sequence of the Mimivirus R555 gene product, originally annotated as a Rad50 homolog, and later shown to have close homologs in marine microbial metagenomes. Results Our bioinformatics analysis revealed that R555 protein sequence is constituted from the fusion of an N-terminal Mre11-like domain with a C-terminal Rad50-like domain. A systematic database search revealed twelve additional cases of Mre11/Rad50 (or SbcD/SbcC fusions in a wide variety of unrelated organisms including unicellular and multicellular eukaryotes, the megaplasmid of a bacterium associated to deep-sea hydrothermal vents (Deferribacter desulfuricans and the plasmid of Clostridium kluyveri. We also showed that R555 homologs are abundant in the metagenomes from different aquatic environments and that they most likely belong to aquatic viruses. The observed phyletic distribution of these fusion proteins suggests their recurrent creation and lateral gene transfers across organisms. Conclusions The existence of the fused version of protein sequences is consistent with known functional interactions between Mre11 and Rad50, and the gene fusion probably enhanced the opportunity for lateral transfer. The abundance of the Mre11/Rad50 fusion genes in viral metagenomes and their sporadic phyletic distribution in cellular organisms suggest that viruses, plasmids and transposons played a crucial role in the formation of the fusion proteins and their propagation into cellular genomes.

  2. A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

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    Yongqian Zhao

    2015-03-01

    Full Text Available Flavivirus RNA replication occurs within a replication complex (RC that assembles on ER membranes and comprises both non-structural (NS viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase and C-terminal RNA-dependent-RNA polymerase (RdRp domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3 at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV, the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

  3. Low immunoglobulin A levels detected via the tissue transglutaminase assay can reveal previously undetected monoclonal proteins.

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    Wallage, M; Dutton, D; Lock, R J

    2016-05-01

    Increased awareness of coeliac disease and the 2009 NICE guidance has led to an increase in patients being screened for Immunoglobulin A deficiency. We have shown previously that this provides an opportunity for the early identification of other underlying primary immunodeficiency, e.g. common variable immunodeficiency. In this context, the underlying gastrointestinal problem appears to be related to bacterial overgrowth. Here, we demonstrate that in addition this also provides an opportunity to reveal underlying secondary immunodeficiency due to other causes in patients with gastrointestinal presentation, notably lymphoproliferative disorders. In one 3-month period, of 60 cases reviewed for low Immunoglobulin A, we found four new paraproteins through this testing route; one symptomatic multiple myeloma, one asymptomatic multiple myeloma, one monoclonal gammopathy of uncertain significance and one in a known chronic lymphocytic leukaemia patient. PMID:26684021

  4. Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life.

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    Niels Lynnerup

    Full Text Available BACKGROUND: Lens crystallines are special proteins in the eye lens. Because the epithelial basement membrane (lens capsule completely encloses the lens, desquamation of aging cells is impossible, and due to the complete absence of blood vessels or transport of metabolites in this area, there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its (14C content from the atmospheric carbon dioxide. The (14C content of the lens proteins thus reflects the atmospheric content of (14C when the lens crystallines were formed. Precise radiocarbon dating is made possible by comparing the (14C content of the lens crystallines to the so-called bomb pulse, i.e. a plot of the atmospheric (14C content since the Second World War, when there was a significant increase due to nuclear-bomb testing. Since the change in concentration is significant even on a yearly basis this allows very accurate dating. METHODOLOGY/PRINCIPAL FINDINGS: Our results allow us to conclude that the crystalline formation in the lens nucleus almost entirely takes place around the time of birth, with a very small, and decreasing, continuous formation throughout life. The close relationship may be further expressed as a mathematical model, which takes into account the timing of the crystalline formation. CONCLUSIONS/SIGNIFICANCE: Such a life-long permanence of human tissue has hitherto only been described for dental enamel. In confront to dental enamel it must be held in mind that the eye lens is a soft structure, subjected to almost continuous deformation, due to lens accommodation, yet its most important constituent, the lens crystalline, is never subject to turnover or remodelling once formed. The determination of the (14C content of various tissues may be used to assess turnover rates and degree of substitution (for example for brain cell DNA. Potential targets may be nervous tissues in terms of senile or pre

  5. A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma.

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    Erin Garrison

    Full Text Available Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle.

  6. Ensemble Activation of G-Protein -Coupled Receptors Revealed by Small-Angle Neutron Scattering

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    Chu, Xiang-Qiang; Perera, Suchithranga; Shrestha, Utsab; Chawla, Udeep; Struts, Andrey; Qian, Shuo; Brown, Michael

    2014-03-01

    Rhodopsin is a G-protein -coupled receptor (GPCR) involved in visual light perception and occurs naturally in a membrane lipid environment. Rhodopsin photoactivation yields cis-trans isomerization of retinal giving equilibrium between inactive Meta-I and active Meta-II states. Does photoactivation lead to a single Meta-II conformation, or do substates exist as described by an ensemble-activation mechanism (EAM)? We use small-angle neutron scattering (SANS) to investigate conformational changes in rhodopsin-detergent and rhodopsin-lipid complexes upon photoactivation. Meta-I state is stabilized in CHAPS-solubilized rhodopsin, while Meta-II is trapped in DDM-solubilized rhodopsin. SANS data are acquired from 80% D2O solutions and at contrast-matching points for both DDM and CHAPS samples. Our experiments demonstrate that for detergent-solubilized rhodopsin, SANS with contrast variation can detect structural differences between the rhodopsin dark-state, Meta-I, Meta-II, and ligand-free opsin states. Dark-state rhodopsin has more conformational flexibility in DDM micelles compared to CHAPS, which is consistent with an ensemble of activated Meta-II states. Furthermore, time-resolved SANS enables study of the time-dependent structural transitions between Meta-I and Meta-II, which is crucial to understanding the ensemble-based activation.

  7. Novel monoclonal antibodies against Pdx1 reveal feedback regulation of Pdx1 protein levels

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    T. Galbo

    2010-04-01

    Full Text Available The aim of this study was to characterize two monoclonal antibodies (F6A11 and F109-D12 generated against Pdx1 (pancreatic and duodenal homeobox-1, a homeodomain transcription factor, which is critical for pancreas formation as well as for normal pancreatic beta cell function. For production of monoclonal antibodies, we immunized Robertsonian POSF (RBFmice with a GST-Pdx1 fusion protein containing a 68-amino acid C-terminal fragment of rat Pdx1. These monoclonal antibodies detect Pdx1 by western blotting and allow immunohistochemical detection of Pdx1 in both mouse and rat tissue. F6A11 and F109-D12 produce IHC staining patterns indistinguishable from that obtained with highly specific polyclonal Pdx1 antisera raised in rabbits and goats, when applied to embryonic or adult mouse pancreatic tissue. In contrast to previously generated polyclonal anti-Pdx1 antisera, we also demonstrate that F6A11 works for intracellular fluorescence activated cell sorting (FACS staining of Pdx1. By using F6A11, we characterize the induction of Pdx1 in the Doxycycline (DOX inducible insulinoma cell line INSrαβ-Pdx1 and follow the reduction of Pdx1 after removing Dox. Finally, we show that induction of exogenous Pdx1 leads to a reduction in endogenous Pdx1 levels, which suggests that a negative feedback loop is involved in maintaining correct levels of Pdx1 in the cell.

  8. Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins.

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    Lavy, Meirav; Prigge, Michael J; Tao, Sibo; Shain, Stephanie; Kuo, April; Kirchsteiger, Kerstin; Estelle, Mark

    2016-01-01

    The coordinated action of the auxin-sensitive Aux/IAA transcriptional repressors and ARF transcription factors produces complex gene-regulatory networks in plants. Despite their importance, our knowledge of these two protein families is largely based on analysis of stabilized forms of the Aux/IAAs, and studies of a subgroup of ARFs that function as transcriptional activators. To understand how auxin regulates gene expression we generated a Physcomitrella patens line that completely lacks Aux/IAAs. Loss of the repressors causes massive changes in transcription with misregulation of over a third of the annotated genes. Further, we find that the aux/iaa mutant is blind to auxin indicating that auxin regulation of transcription occurs exclusively through Aux/IAA function. We used the aux/iaa mutant as a simplified platform for studies of ARF function and demonstrate that repressing ARFs regulate auxin-induced genes and fine-tune their expression. Further the repressing ARFs coordinate gene induction jointly with activating ARFs and the Aux/IAAs. PMID:27247276

  9. Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins

    Science.gov (United States)

    Lavy, Meirav; Prigge, Michael J; Tao, Sibo; Shain, Stephanie; Kuo, April; Kirchsteiger, Kerstin; Estelle, Mark

    2016-01-01

    The coordinated action of the auxin-sensitive Aux/IAA transcriptional repressors and ARF transcription factors produces complex gene-regulatory networks in plants. Despite their importance, our knowledge of these two protein families is largely based on analysis of stabilized forms of the Aux/IAAs, and studies of a subgroup of ARFs that function as transcriptional activators. To understand how auxin regulates gene expression we generated a Physcomitrella patens line that completely lacks Aux/IAAs. Loss of the repressors causes massive changes in transcription with misregulation of over a third of the annotated genes. Further, we find that the aux/iaa mutant is blind to auxin indicating that auxin regulation of transcription occurs exclusively through Aux/IAA function. We used the aux/iaa mutant as a simplified platform for studies of ARF function and demonstrate that repressing ARFs regulate auxin-induced genes and fine-tune their expression. Further the repressing ARFs coordinate gene induction jointly with activating ARFs and the Aux/IAAs. DOI: http://dx.doi.org/10.7554/eLife.13325.001 PMID:27247276

  10. Dynamic features of apo and bound HIV-Nef protein reveal the anti-HIV dimerization inhibition mechanism.

    Science.gov (United States)

    Moonsamy, Suri; Bhakat, Soumendranath; Soliman, Mahmoud E S

    2015-01-01

    The first account on the dynamic features of Nef or negative factor, a small myristoylated protein located in the cytoplasm believes to increase HIV-1 viral titer level, is reported herein. Due to its major role in HIV-1 pathogenicity, Nef protein is considered an emerging target in anti-HIV drug design and discovery process. In this study, comparative long-range all-atom molecular dynamics simulations were employed for apo and bound protein to unveil molecular mechanism of HIV-Nef dimerization and inhibition. Results clearly revealed that B9, a newly discovered Nef inhibitor, binds at the dimeric interface of Nef protein and caused significant separation between orthogonally opposed residues, namely Asp108, Leu112 and Gln104. Large differences in magnitudes were observed in the radius of gyration (∼1.5 Å), per-residue fluctuation (∼2 Å), C-alpha deviations (∼2 Å) which confirm a comparatively more flexible nature of apo conformation due to rapid dimeric association. Compared to the bound conformer, a more globally correlated motion in case of apo structure of HIV-Nef confirms the process of dimeric association. This clearly highlights the process of inhibition as a result of ligand binding. The difference in principal component analysis (PCA) scatter plot and per-residue mobility plot across first two normal modes further justifies the same findings. The in-depth dynamic analyses of Nef protein presented in this report would serve crucial in understanding its function and inhibition mechanisms. Information on inhibitor binding mode would also assist in designing of potential inhibitors against this important HIV target. PMID:26355431

  11. The role of negative selection in protein evolution revealed through the energetics of the native state ensemble.

    Science.gov (United States)

    Hoffmann, Jordan; Wrabl, James O; Hilser, Vincent J

    2016-04-01

    Knowing the determinants of conformational specificity is essential for understanding protein structure, stability, and fold evolution. To address this issue, a novel statistical measure of energetic compatibility between sequence and structure was developed using an experimentally validated model of the energetics of the native state ensemble. This approach successfully matched sequences from a diverse subset of the human proteome to their respective folds. Unexpectedly, significant energetic compatibility between ostensibly unrelated sequences and structures was also observed. Interrogation of these matches revealed a general framework for understanding the origins of conformational specificity within a proteome: specificity is a complex function of both the ability of a sequence to adopt folds other than the native, and ability of a fold to accommodate sequences other than the native. The regional variation in energetic compatibility indicates that the compatibility is dominated by incompatibility of sequence for alternative fold segments, suggesting that evolution of protein sequences has involved substantial negative selection, with certain segments serving as "gatekeepers" that presumably prevent alternative structures. Beyond these global trends, a size dependence exists in the degree to which the energetic compatibility is determined from negative selection, with smaller proteins displaying more negative selection. This partially explains how short sequences can adopt unique folds, despite the higher probability in shorter proteins for small numbers of mutations to increase compatibility with other folds. In providing evolutionary ground rules for the thermodynamic relationship between sequence and fold, this framework imparts valuable insight for rational design of unique folds or fold switches. Proteins 2016; 84:435-447. © 2016 Wiley Periodicals, Inc. PMID:26800099

  12. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

    Energy Technology Data Exchange (ETDEWEB)

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M.F.; Downes, C. Peter; Batty, Ian H. (Toronto); (Dundee)

    2012-10-16

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

  13. Small Molecule Inhibitors of Ca(2+)-S100B Reveal Two Protein Conformations.

    Science.gov (United States)

    Cavalier, Michael C; Ansari, Mohd Imran; Pierce, Adam D; Wilder, Paul T; McKnight, Laura E; Raman, E Prabhu; Neau, David B; Bezawada, Padmavani; Alasady, Milad J; Charpentier, Thomas H; Varney, Kristen M; Toth, Eric A; MacKerell, Alexander D; Coop, Andrew; Weber, David J

    2016-01-28

    The drug pentamidine inhibits calcium-dependent complex formation with p53 ((Ca)S100B·p53) in malignant melanoma (MM) and restores p53 tumor suppressor activity in vivo. However, off-target effects associated with this drug were problematic in MM patients. Structure-activity relationship (SAR) studies were therefore completed here with 23 pentamidine analogues, and X-ray structures of (Ca)S100B·inhibitor complexes revealed that the C-terminus of S100B adopts two different conformations, with location of Phe87 and Phe88 being the distinguishing feature and termed the "FF-gate". For symmetric pentamidine analogues ((Ca)S100B·5a, (Ca)S100B·6b) a channel between sites 1 and 2 on S100B was occluded by residue Phe88, but for an asymmetric pentamidine analogue ((Ca)S100B·17), this same channel was open. The (Ca)S100B·17 structure illustrates, for the first time, a pentamidine analog capable of binding the "open" form of the "FF-gate" and provides a means to block all three "hot spots" on (Ca)S100B, which will impact next generation (Ca)S100B·p53 inhibitor design. PMID:26727270

  14. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles

    Science.gov (United States)

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  15. Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants.

    Science.gov (United States)

    Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Choi, HyungWon; Sobreira, Tiago J P; Nohara, Lilian L; Wermelinger, Luciana S; Almeida, Igor C; Zingali, Russolina B; Fernandes, Patricia M B

    2012-06-18

    Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses. PMID:22465191

  16. Proteome profiling of the growth phases of Leishmania pifanoi promastigotes in axenic culture reveals differential abundance of immunostimulatory proteins.

    Science.gov (United States)

    Alcolea, Pedro J; Alonso, Ana; García-Tabares, Francisco; Mena, María del Carmen; Ciordia, Sergio; Larraga, Vicente

    2016-06-01

    Leishmaniasis is a term that encompasses a compendium of neglected tropical diseases caused by dimorphic and digenetic protozoan parasites from the genus Leishmania (Kinetoplastida: Trypanosomatidae). The clinical manifestations of neotropical cutaneous leishmaniasis (NCL) caused by Leishmania pifanoi and other species of the "Leishmania mexicana complex" mainly correspond to anergic diffuse cutaneous leishmaniasis (ADCL), which is the origin of considerable morbidity. Despite the outstanding advances in the characterization of the trypanosomatid genomes and proteomes, the biology of this species has been scarcely explored. However, the close relation of L. pifanoi to the sequenced species L. mexicana and others included in the "L. mexicana complex" allowed us to perform a two-dimension electrophoresis (2DE) approach to the promastigote proteome at the differential expression level. Protein identifications were performed by matrix-assisted laser desorption-ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). This insight has revealed similarities and differences between L. pifanoi and other species responsible for cutaneous and visceral leishmaniasis. Interestingly, certain proteins that were previously described as immunostimulatory (elongation factor 1β, trypanothione peroxidase, heat shock protein 70, enolase, GDP-forming succinyl-CoA and aldehyde dehydrogenase) are more abundant in the final growth stages of promastigotes (late-logarithmic and/or stationary phase) in the case of L. pifanoi. PMID:26992294

  17. A crayfish insulin-like-binding protein: another piece in the androgenic gland insulin-like hormone puzzle is revealed.

    Science.gov (United States)

    Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir

    2013-08-01

    Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a "pulldown" methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23-38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079

  18. A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2.

    Science.gov (United States)

    Breitkopf, Susanne B; Yang, Xuemei; Begley, Michael J; Kulkarni, Meghana; Chiu, Yu-Hsin; Turke, Alexa B; Lauriol, Jessica; Yuan, Min; Qi, Jie; Engelman, Jeffrey A; Hong, Pengyu; Kontaridis, Maria I; Cantley, Lewis C; Perrimon, Norbert; Asara, John M

    2016-01-01

    Using a series of immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway. PMID:26839216

  19. A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2

    Science.gov (United States)

    Breitkopf, Susanne B.; Yang, Xuemei; Begley, Michael J.; Kulkarni, Meghana; Chiu, Yu-Hsin; Turke, Alexa B.; Lauriol, Jessica; Yuan, Min; Qi, Jie; Engelman, Jeffrey A.; Hong, Pengyu; Kontaridis, Maria I.; Cantley, Lewis C.; Perrimon, Norbert; Asara, John M.

    2016-02-01

    Using a series of immunoprecipitation (IP) - tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

  20. Kinetic and Conformational Insights of Protein Adsorption onto Montmorillonite Revealed Using in Situ ATR-FTIR/2D-COS.

    Science.gov (United States)

    Schmidt, Michael P; Martínez, Carmen Enid

    2016-08-01

    Protein adsorption onto clay minerals is a process with wide-ranging impacts on the environmental cycling of nutrients and contaminants. This process is influenced by kinetic and conformational factors that are often challenging to probe in situ. This study represents an in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopic investigation of the adsorption of a model protein (bovine serum albumin (BSA)) onto a clay mineral (montmorillonite) at four concentrations (1.50, 3.75, 7.50, and 15.0 μM) under environmentally relevant conditions. At all concentrations probed, FTIR spectra show that BSA readily adsorbs onto montmorillonite. Adsorption kinetics follow an Elovich model, suggesting that primary limitations on adsorption rates are surface-related heterogeneous energetic restrictions associated with protein rearrangement and lateral protein-protein interaction. BSA adsorption onto montmorillonite fits the Langmuir model, yielding K = 5.97 × 10(5) M(-1). Deconvolution and curve fitting of the amide I band at the end of the adsorption process (∼120 min) shows a large extent of BSA unfolding upon adsorption at 1.50 μM, with extended chains and turns increasing at the expense of α-helices. At higher concentrations/surface coverages, BSA unfolding is less pronounced and a more compact structure is assumed. Two-dimensional correlation spectroscopic (2D-COS) analysis reveals three different pathways corresponding to adsorbed conformations. At 1.50 μM, adsorption increases extended chains, followed by a loss in α-helices and a subsequent increase in turns. At 3.75 μM, extended chains decrease and then aggregated strands increase and side chains decrease, followed by a decrease in turns. With 7.50 and 15.0 μM BSA, the loss of side-chain vibrations is followed by an increase in aggregated strands and a subsequent decrease in turns and extended chains. Overall, the BSA concentration and resultant surface coverage have a profound

  1. Comparative Proteome Analysis of the Tuberous Roots of Six Cassava (Manihot esculenta) Varieties Reveals Proteins Related to Phenotypic Traits.

    Science.gov (United States)

    Schmitz, Gabriela Justamante Händel; de Magalhães Andrade, Jonathan; Valle, Teresa Losada; Labate, Carlos Alberto; do Nascimento, João Roberto Oliveira

    2016-04-27

    Cassava (Manihot esculenta Crantz) is a staple food and an important source of starch, and the attributes of its tuberous root largely depend on the variety. The proteome of cassava has been investigated; however, to date, no study has focused on varieties that reveal the molecular basis of phenotypical characteristics. Therefore, we aimed to compare the proteome of the tuberous roots of six cassava varieties that differed in carbohydrates, carotenoids, and resistance to diseases, among other attributes. Two-dimensional gels showed 146 differential spots between the varieties, and the functional roles of some differential proteins were correlated to phenotypic characteristics of the varieties, such as the amount of carbohydrates or carotenoids and the resistance to biotic or abiotic stresses. The results obtained here highlight elements that might help to direct the improvement of new cultivars of cassava, which is an economically and socially relevant crop worldwide. PMID:26982619

  2. Differential Protein Expression in Sugarcane during Sugarcane-Sporisorium scitamineum Interaction Revealed by 2-DE and MALDI-TOF-TOF/MS

    OpenAIRE

    Youxiong Que; Liping Xu; Jianwei Lin; Miaohong Ruan; Muqing Zhang; Rukai Chen

    2011-01-01

    To understand the molecular basis of a specific plant-pathogen interaction, it is important to identify plant proteins that respond to the pathogen attack. Two sugarcane varieties, NCo376 and Ya71-374, were used in this study. By applying 2-dimensional electrophoresis (2-DE), the protein expression profile of sugarcane after inoculating with Sporisorium scitamineum was analyzed. In total, 23 differentially expressed proteins were identified by MALDI-TOF-TOF/MS. Bioinformatics analysis reveale...

  3. Clonal mosaic analysis of EMPTY PERICARP2 reveals nonredundant functions of the duplicated HEAT SHOCK FACTOR BINDING PROTEINs during maize shoot development.

    OpenAIRE

    Fu, Suneng; Scanlon, Michael J.

    2004-01-01

    The paralogous maize proteins EMPTY PERICARP2 (EMP2) and HEAT SHOCK FACTOR BINDING PROTEIN2 (HSBP2) each contain a single recognizable motif: the coiled-coil domain. EMP2 and HSBP2 accumulate differentially during maize development and heat stress. Previous analyses revealed that EMP2 is required for regulation of heat shock protein (hsp) gene expression and also for embryo morphogenesis. Developmentally abnormal emp2 mutant embryos are aborted during early embryogenesis. To analyze EMP2 func...

  4. Proteomic analysis of pig (Sus scrofa olfactory soluble proteome reveals O-GlcNAcylation of secreted odorant-binding proteins

    Directory of Open Access Journals (Sweden)

    Patricia eNAGNAN-LE MEILLOUR

    2014-12-01

    Full Text Available The diversity of olfactory binding proteins (OBPs is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

  5. Structures of Rhodopsin Kinase in Different Ligand States Reveal Key Elements Involved in G Protein-coupled Receptor Kinase Activation

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Puja; Wang, Benlian; Maeda, Tadao; Palczewski, Krzysztof; Tesmer, John J.G. (Case Western); (Michigan)

    2008-10-08

    G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins. Here we report six crystal structures of rhodopsin kinase (GRK1), revealing not only three distinct nucleotide-binding states of a GRK but also two key structural elements believed to be involved in the recognition of activated GPCRs. The first is the C-terminal extension of the kinase domain, which was observed in all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N terminus, observed in one of the GRK1{center_dot}(Mg{sup 2+}){sub 2} {center_dot}ATP structures. The N terminus was also clearly phosphorylated, leading to the identification of two novel phosphorylation sites by mass spectral analysis. Co-localization of the N terminus and the C-terminal extension near the hinge of the kinase domain suggests that activated GPCRs stimulate kinase activity by binding to this region to facilitate full closure of the kinase domain.

  6. Comparative Analysis of JmjC Domain-containing Proteins Reveals the Potential Histone Demethylases in Arabidopsis and Rice

    Institute of Scientific and Technical Information of China (English)

    Falong Lu; Guanglin Li; Xia Cui; Chunyan Liu; Xiu-Jie Wang; Xiaofeng Cao

    2008-01-01

    Histone methylation homeostasis is achieved by controlling the balance between methylation and demethylation to maintain chromatin function and developmental regulation. In animals, a conserved Jumonji C (JmjC) domain was found In a large group of histone demethylases. However, it is still unclear whether plants also contain the JmjC domaincontaining active histone demethylases. Here we performed genome-wide screen and phylogenetic analysis of JmjC domaincontaining proteins in the dicot plant, Arabidopsis, and monocot plant rice, and found 21 and 20 JmjC domain-containing, respectively. We also examined the expression of JmjC domain-containing proteins and compared them to human JmjC counterparts for potential enzymatic activity. The spatial expression patterns of the Arabidopsis JmjC domaincontaining genes revealed that they are all actively transcribed genes. These active plant JmjC domain-containing genes could possibly function in epigenetic regulation to antagonize the activity of the large number of putative SET domaincontaining histone methyltransferase activity to dynamically regulate histone methylation homeostasis.

  7. Microwave & Magnetic (M2) Proteomics Reveals CNS-Specific Protein Expression Waves that Precede Clinical Symptoms of Experimental Autoimmune Encephalomyelitis

    Science.gov (United States)

    Raphael, Itay; Mahesula, Swetha; Purkar, Anjali; Black, David; Catala, Alexis; Gelfond, Jonathon A. L.; Forsthuber, Thomas G.; Haskins, William E.

    2014-09-01

    Central nervous system-specific proteins (CSPs), transported across the damaged blood-brain-barrier (BBB) to cerebrospinal fluid (CSF) and blood (serum), might be promising diagnostic, prognostic and predictive protein biomarkers of disease in individual multiple sclerosis (MS) patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. We hypothesized that microwave & magnetic (M2) proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in serum. To test this hypothesis, we used M2 proteomics to longitudinally assess CSP expression in brain tissue from mice during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Confirmation of central nervous system (CNS)-infiltrating inflammatory cell response and CSP expression in serum was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. M2 proteomics (and ELISA) revealed characteristic CSP expression waves, including synapsin-1 and α-II-spectrin, which peaked at day 7 in brain tissue (and serum) and preceded clinical EAE symptoms that began at day 10 and peaked at day 20. Moreover, M2 proteomics supports the concept that relatively few CNS-infiltrating inflammatory cells can have a disproportionally large impact on CSP expression prior to clinical manifestation of EAE.

  8. Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.

    Directory of Open Access Journals (Sweden)

    Colin P De Souza

    Full Text Available Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs, the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN specifically at SPBs in the basal region of G1 cells and that

  9. Triclosan Resistome from Metagenome Reveals Diverse Enoyl Acyl Carrier Protein Reductases and Selective Enrichment of Triclosan Resistance Genes.

    Science.gov (United States)

    Khan, Raees; Kong, Hyun Gi; Jung, Yong-Hoon; Choi, Jinhee; Baek, Kwang-Yeol; Hwang, Eul Chul; Lee, Seon-Woo

    2016-01-01

    Triclosan (TCS) is a widely used antimicrobial agent and TCS resistance is considered to have evolved in diverse organisms with extensive use of TCS, but distribution of TCS resistance has not been well characterized. Functional screening of the soil metagenome in this study has revealed that a variety of target enoyl acyl carrier protein reductases (ENR) homologues are responsible for the majority of TCS resistance. Diverse ENRs similar to 7-α-hydroxysteroid dehydrogenase (7-α-HSDH), FabG, or the unusual YX7K-type ENR conferred extreme tolerance to TCS. The TCS-refractory 7-α HSDH-like ENR and the TCS-resistant YX7K-type ENR seem to be prevalent in human pathogenic bacteria, suggesting that a selective enrichment occurred in pathogenic bacteria in soil. Additionally, resistance to multiple antibiotics was found to be mediated by antibiotic resistance genes that co-localize with TCS resistance determinants. Further comparative analysis of ENRs from 13 different environments has revealed a huge diversity of both prototypic and metagenomic TCS-resistant ENRs, in addition to a selective enrichment of TCS-resistant specific ENRs in presumably TCS-contaminated environments with reduced ENR diversity. Our results suggest that long-term extensive use of TCS can lead to the selective emergence of TCS-resistant bacterial pathogens, possibly with additional resistance to multiple antibiotics, in natural environments. PMID:27577999

  10. Triclosan Resistome from Metagenome Reveals Diverse Enoyl Acyl Carrier Protein Reductases and Selective Enrichment of Triclosan Resistance Genes

    Science.gov (United States)

    Khan, Raees; Kong, Hyun Gi; Jung, Yong-Hoon; Choi, Jinhee; Baek, Kwang-Yeol; Hwang, Eul Chul; Lee, Seon-Woo

    2016-01-01

    Triclosan (TCS) is a widely used antimicrobial agent and TCS resistance is considered to have evolved in diverse organisms with extensive use of TCS, but distribution of TCS resistance has not been well characterized. Functional screening of the soil metagenome in this study has revealed that a variety of target enoyl acyl carrier protein reductases (ENR) homologues are responsible for the majority of TCS resistance. Diverse ENRs similar to 7-α-hydroxysteroid dehydrogenase (7-α-HSDH), FabG, or the unusual YX7K-type ENR conferred extreme tolerance to TCS. The TCS-refractory 7-α HSDH-like ENR and the TCS-resistant YX7K-type ENR seem to be prevalent in human pathogenic bacteria, suggesting that a selective enrichment occurred in pathogenic bacteria in soil. Additionally, resistance to multiple antibiotics was found to be mediated by antibiotic resistance genes that co-localize with TCS resistance determinants. Further comparative analysis of ENRs from 13 different environments has revealed a huge diversity of both prototypic and metagenomic TCS-resistant ENRs, in addition to a selective enrichment of TCS-resistant specific ENRs in presumably TCS-contaminated environments with reduced ENR diversity. Our results suggest that long-term extensive use of TCS can lead to the selective emergence of TCS-resistant bacterial pathogens, possibly with additional resistance to multiple antibiotics, in natural environments. PMID:27577999

  11. Structures of Neuroligin-1 And the Neuroligin-1/Neurexin-1beta Complex Reveal Specific Protein-Protein And Protein-Ca**2+ Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Arac, D.; Boucard, A.A.; Ozkan, E.A; Strop, P.; Newell, E.; Sudhof, T.C.; Brunger, A.T.

    2009-05-28

    Neurexins and neuroligins provide trans-synaptic connectivity by the Ca{sup 2+}-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1{beta}. Neuroligin-1 forms a constitutive dimer, and two neurexin-1{beta} monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1{beta} complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca{sup 2+}. Alternatively spliced sites in neurexin-1{beta} and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1{beta}, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1{beta} complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

  12. Structures of Neuroligin-1 And the Neuroligin-1/Neurexin-1 Beta Complex Reveal Specific Protein-Protein And Protein-Ca2+ Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Demet, Arac; Boucard, A.A.; Ozkan, E.A; Strop, P.; Newell, E.; Sudhof, T.C.; Brunger, A.T.

    2009-06-01

    Neurexins and neuroligins provide trans-synaptic connectivity by the Ca{sup 2+}-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1{beta}. Neuroligin-1 forms a constitutive dimer, and two neurexin-1{beta} monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1{beta} complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca{sup 2+}. Alternatively spliced sites in neurexin-1{beta} and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1{beta}, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1{beta} complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

  13. A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.

    Science.gov (United States)

    Dedic, Emil; Alsarraf, Husam; Welner, Ditte Hededam; Østergaard, Ole; Klychnikov, Oleg I; Hensbergen, Paul J; Corver, Jeroen; van Leeuwen, Hans C; Jørgensen, René

    2016-06-17

    Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP γ-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the γ-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory α-helix. PMID:27076635

  14. Revealing the mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan binding using molecular simulation

    Directory of Open Access Journals (Sweden)

    Olgun eGuvench

    2015-06-01

    Full Text Available The extracellular N-terminal hyaluronan binding domain (HABD of CD44 is a small globular domain that confers hyaluronan (HA binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-soluble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, and such glycosylation can diminish HA binding when the associated N-glycans are capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of N-glycans makes it difficult to apply experimental structural biology approaches to probe the molecular mechanisms of how the order-to-disorder transition and N-glycosylation can modulate HA binding by HABD. We review recent results from molecular dynamics simulations that provide atomic-resolution mechanistic understanding of such modulation to help bridge gaps between existing experimental binding and structural biology data. Findings from these simulations include: Tyr42 may function as a molecular switch that converts the HA binding site from a low affinity to a high affinity state; in the partially-disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient mobility to form direct contacts with bound HA to further stabilize binding; and terminal sialic acids on covalently-attached N-glycans can form charge-paired hydrogen bonding interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA binding to glycosylated CD44 HABD.

  15. Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.

    Science.gov (United States)

    Cole, Daniel J; Rajendra, Eeson; Roberts-Thomson, Meredith; Hardwick, Bryn; McKenzie, Grahame J; Payne, Mike C; Venkitaraman, Ashok R; Skylaris, Chris-Kriton

    2011-07-01

    The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the

  16. Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.

    Directory of Open Access Journals (Sweden)

    Daniel J Cole

    2011-07-01

    Full Text Available The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and

  17. Proteome profiling reveals tissue-specific protein expression in male and female accessory glands of the silkworm, Bombyx mori.

    Science.gov (United States)

    Dong, Zhaoming; Wang, Xiaohuan; Zhang, Yan; Zhang, Liping; Chen, Quanmei; Zhang, Xiaolu; Zhao, Ping; Xia, Qingyou

    2016-05-01

    Male accessory gland (MAG) and female accessory gland (FAG) of the reproductive system are, respectively, responsible for producing seminal proteins and adhesive proteins during copulation and ovulation. Seminal proteins are ejaculated to female along with sperms, whereas adhesive proteins are excreted along with eggs. Proteins from the male and female reproductive organs are usually indicative of rapid adaptive evolution. Understanding the reproductive isolation and species divergence requires identifying reproduction-related proteins from many different species. Here, we present our proteomic analyses of male and female accessory glands of the silkworm, Bombyx mori. Using LC/MS-MS, we identified 2133 MAG proteins and 1872 FAG proteins. In total, 652 proteins were significant more abundant in the MAG than in the FAG, including growth factors, odorant-binding proteins, enzymes, and proteins of unknown function. Growth factors and odorant-binding proteins are potential signaling molecules, whereas most of proteins of unknown function were found to be Lepidoptera-specific proteins with high evolutionary rates. Microarray experiments and semi-quantitative RT-PCR validated that MAG-specific proteins were expressed exclusively in male moths. Totally, 192 proteins were considered as FAG-specific proteins, including protease inhibitors, enzymes, and other proteins. Protease inhibitors were found to be the most abundant FAG-specific proteins, which may protect eggs from infection by inhibiting pathogen-derived proteases. These results provide comprehensive insights into copulation and oviposition. Moreover, the newly identified Lepidoptera-specific MAG proteins provide useful data for future research on the evolution of reproductive proteins in insects. PMID:26822097

  18. Loss of Arabidopsis thaliana Dynamin-Related Protein 2B reveals separation of innate immune signaling pathways.

    Directory of Open Access Journals (Sweden)

    John M Smith

    2014-12-01

    Full Text Available Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B, which has been previously implicated in constitutive clathrin-mediated endocytosis (CME, functions in responses to flg22 (the active peptide derivative of bacterial flagellin and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI, drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC-, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD, the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca2+-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC-. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2, was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects

  19. Dietary soyasaponin supplementation to pea protein concentrate reveals nutrigenomic interactions underlying enteropathy in Atlantic salmon (Salmo salar

    Directory of Open Access Journals (Sweden)

    Kortner Trond M

    2012-07-01

    Full Text Available Abstract Background Use of plant ingredients in aquaculture feeds is impeded by high contents of antinutritional factors such as saponins, which may cause various pharmacological and biological effects. In this study, transcriptome changes were analyzed using a 21 k oligonucleotide microarray and qPCR in the distal intestine of Atlantic salmon fed diets based on five plant protein sources combined with soybean saponins. Results Diets with corn gluten, sunflower, rapeseed or horsebean produced minor effects while the combination of saponins with pea protein concentrate caused enteritis and major transcriptome changes. Acute inflammation was characterised by up-regulation of cytokines, NFkB and TNFalpha related genes and regulators of T-cell function, while the IFN-axis was suppressed. Induction of lectins, complement, metalloproteinases and the respiratory burst complex parallelled a down-regulation of genes for free radical scavengers and iron binding proteins. Marked down-regulation of xenobiotic metabolism was also observed, possibly increasing vulnerability of the intestinal tissue. A hallmark of metabolic changes was dramatic down-regulation of lipid, bile and steroid metabolism. Impairment of digestion was further suggested by expression changes of nutrient transporters and regulators of water balance (e.g. aquaporin, guanylin. On the other hand, microarray profiling revealed activation of multiple mucosal defence processes. Annexin-1, with important anti-inflammatory and gastroprotective properties, was markedly up-regulated. Furthermore, augmented synthesis of polyamines needed for cellular proliferation (up-regulation of arginase and ornithine decarboxylase and increased mucus production (down-regulation of glycan turnover and goblet cell hyperplasia could participate in mucosal healing and restoration of normal tissue function. Conclusion The current study promoted understanding of salmon intestinal pathology and establishment of a

  20. The extracellular proteome of Bifidobacterium animalis subsp. lactis BB‐12 reveals proteins with putative roles in probiotic effects

    DEFF Research Database (Denmark)

    Gilad, Ofir; Svensson, Birte; Viborg, Alexander Holm; Stuer‐Lauridsen, Birgitte; Jacobsen, Susanne

    2011-01-01

    Bifidobacterium animalis subsp. lactis BB‐12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2‐DE coupled with MALDI‐TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either...... outside the cell or on its surface. These proteins include solute‐binding proteins for oligosaccharides, amino acids and manganese, cell wall‐metabolizing proteins, and 18 proteins that have been described to interact with human host epithelial cells or extracellular matrix proteins. The potential...

  1. Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics

    OpenAIRE

    Smits, A. H.; Jansen, P. W. T. C.; Poser, I; Hyman, A. A.; Vermeulen, M.

    2013-01-01

    Many cellular proteins assemble into macromolecular protein complexes. The identification of protein–protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not sui...

  2. Molecular basis of photochromism of a fluorescent protein revealed by direct 13C detection under laser illumination

    International Nuclear Information System (INIS)

    Dronpa is a green fluorescent protein homologue with a photochromic property. A green laser illumination reversibly converts Dronpa from a green-emissive bright state to a non-emissive dark state, and ultraviolet illumination converts it to the bright state. We have employed solution NMR to understand the underlying molecular mechanism of the photochromism. The detail characterization of Dronpa is hindered as it is metastable in the dark state and spontaneously converts to the bright state. To circumvent this issue, we have designed in magnet laser illumination device. By combining the device with a 150-mW argon laser at 514.5 nm, we have successfully converted and maintained Dronpa in the dark state in the NMR tube by continuous illumination during the NMR experiments. We have employed direct-detection of 13C nuclei from the carbon skeleton of the chromophore for detailed characterization of chromophore in both states of Dronpa by using the Bruker TCI cryoprobe. The results from NMR data have provided direct evidence of the double bond formation between Cα and Cβ of Y63 in the chromophore, the β-barrel structure in solution, and the ionized and protonated state of Y63 hydroxyl group in the bright and dark states, respectively. These studies have also revealed that a part of β-barrel around the chromophore becomes polymorphic only in the dark state, which may be critical to make the fluorescence dim by increasing the contribution of non-emissive vibrational relaxation pathways.

  3. Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    2014-02-01

    Full Text Available Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI screens can provide insights into the biological role(s of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

  4. Dissection of an old protein reveals a novel application: domain D of Staphylococcus aureus Protein A (sSpAD as a secretion - tag

    Directory of Open Access Journals (Sweden)

    Paal Michael

    2010-11-01

    Full Text Available Abstract Background Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous. Results We discovered that domain D of the Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation. The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD. Conclusions In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.

  5. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases

    Energy Technology Data Exchange (ETDEWEB)

    Crosby, Heidi A [University of Wisconsin, Madison; Pelletier, Dale A [ORNL; Hurst, Gregory {Greg} B [ORNL; Escalante-Semerena, Jorge C [University of Wisconsin, Madison

    2012-01-01

    Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

  6. Single molecule force spectroscopy reveals engineered metal chelation is a general approach to enhance mechanical stability of proteins

    OpenAIRE

    Cao, Yi; Yoo, Teri; Li, Hongbin

    2008-01-01

    Significant mechanical stability is an essential feature shared by many elastomeric proteins, which function as molecular springs in a wide variety of biological machinery and biomaterials of superb mechanical properties. Despite the progress in understanding molecular determinants of mechanical stability, it remains challenging to rationally enhance the mechanical stability of proteins. Using single molecule force spectroscopy and protein engineering techniques, we demonstrate that engineere...

  7. Overflow of a hyper-produced secretory protein from the Bacillus Sec pathway into the Tat pathway for protein secretion as revealed by proteogenomics

    NARCIS (Netherlands)

    Kouwen, Thijs R. H. M.; van der Ploeg, Rene; Antelmann, Haike; Hecker, Michael; Homuth, Georg; Maeder, Ulrike; van Dijl, Jan Maarten

    2009-01-01

    Bacteria secrete numerous proteins into their environment for growth and survival under complex and ever-changing conditions. The highly different characteristics of secreted proteins pose major challenges to the cellular protein export machinery and, accordingly, different pathways have evolved. Wh

  8. Microarray analysis of nemorosone-induced cytotoxic effects on pancreatic cancer cells reveals activation of the unfolded protein response (UPR)

    Science.gov (United States)

    Holtrup, Frank; Bauer, Andrea; Fellenberg, Kurt; Hilger, Ralf A; Wink, Michael; Hoheisel, Jörg D

    2011-01-01

    BACKGROUND AND PURPOSE Pancreatic cancer is one of the leading cancer-related causes of death due to high chemo-resistance and fast metastasation. Nemorosone, a polycyclic polyprenylated acylphloroglucinol, has recently been identified as a promising anticancer agent. Here, we examine its growth-inhibitory effects on pancreatic cancer cells. Based on transcription profiling, a molecular mode of action is proposed. EXPERIMENTAL APPROACH Nemorosone cytotoxicity was assessed by the resazurin proliferation assay on pancreatic cancer cells and fibroblasts. Apoptosis was determined by Annexin V/propidium iodide staining as well as cytochrome c and caspase activation assays. Staining with the voltage-dependent dye JC-1 and fluorescence microscopy were used to detect effects on mitochondrial membrane potential. Total RNA was isolated from treated cell lines and subjected to microarray analysis, subsequent pathway identification and modelling. Gene expression data were validated by quantitative polymerase chain reaction and siRNA-mediated gene knock-down. KEY RESULTS Nemorosone significantly inhibited cancer cell growth, induced cytochrome c release and subsequent caspase-dependent apoptosis, rapidly abolished mitochondrial membrane potential and elevated cytosolic calcium levels, while fibroblasts were largely unaffected. Expression profiling revealed 336 genes to be affected by nemorosone. A total of 75 genes were altered in all three cell lines, many of which were within the unfolded protein response (UPR) network. DNA damage inducible transcript 3 was identified as a key regulator in UPR-mediated cell death. CONCLUSIONS AND IMPLICATIONS Nemorosone could be a lead compound for the development of novel anticancer drugs amplifying the already elevated UPR level in solid tumours, thus driving them into apoptosis. This study forms the basis for further investigations identifying nemorosone's direct molecular target(s). PMID:21091652

  9. Molecular basis of photochromism of a fluorescent protein revealed by direct {sup 13}C detection under laser illumination

    Energy Technology Data Exchange (ETDEWEB)

    Mizuno, Hideaki, E-mail: hideaki.mizuno@chem.kuleuven.be; Mal, Tapas Kumar [Katholieke Universiteit Leuven, Department of Chemistry and INPAC (Belgium); Waelchli, Markus [Bruker BioSpin K.K (Japan); Fukano, Takashi [Brain Science Institute, RIKEN, Cell Function and Dynamics (Japan); Ikura, Mitsuhiko [University of Toronto, MaRS Toronto Medical Dicovery Tower, Division of Signaling Biology, Ontario Cancer Institute and Department of Medical Biophysics (Canada); Miyawaki, Atsushi, E-mail: matsushi@brain.riken.j [Brain Science Institute, RIKEN, Cell Function and Dynamics (Japan)

    2010-12-15

    Dronpa is a green fluorescent protein homologue with a photochromic property. A green laser illumination reversibly converts Dronpa from a green-emissive bright state to a non-emissive dark state, and ultraviolet illumination converts it to the bright state. We have employed solution NMR to understand the underlying molecular mechanism of the photochromism. The detail characterization of Dronpa is hindered as it is metastable in the dark state and spontaneously converts to the bright state. To circumvent this issue, we have designed in magnet laser illumination device. By combining the device with a 150-mW argon laser at 514.5 nm, we have successfully converted and maintained Dronpa in the dark state in the NMR tube by continuous illumination during the NMR experiments. We have employed direct-detection of {sup 13}C nuclei from the carbon skeleton of the chromophore for detailed characterization of chromophore in both states of Dronpa by using the Bruker TCI cryoprobe. The results from NMR data have provided direct evidence of the double bond formation between C{sup {alpha}} and C{sup {beta}} of Y63 in the chromophore, the {beta}-barrel structure in solution, and the ionized and protonated state of Y63 hydroxyl group in the bright and dark states, respectively. These studies have also revealed that a part of {beta}-barrel around the chromophore becomes polymorphic only in the dark state, which may be critical to make the fluorescence dim by increasing the contribution of non-emissive vibrational relaxation pathways.

  10. Sub-terahertz spectroscopy reveals that proteins influence the properties of water at greater distances than previously detected

    Energy Technology Data Exchange (ETDEWEB)

    Sushko, Oleksandr; Dubrovka, Rostyslav; Donnan, Robert S., E-mail: r.donnan@qmul.ac.uk [School of Electronic Engineering and Computer Science, Queen Mary University of London, Mile End Road, London E1 4NS (United Kingdom)

    2015-02-07

    The initial purpose of the study is to systematically investigate the solvation properties of different proteins in water solution by terahertz (THz) radiation absorption. Transmission measurements of protein water solutions have been performed using a vector network analyser-driven quasi-optical bench covering the WR-3 waveguide band (0.220–0.325 THz). The following proteins, ranging from low to high molecular weight, were chosen for this study: lysozyme, myoglobin, and bovine serum albumin (BSA). Absorption properties of solutions were studied at different concentrations of proteins ranging from 2 to 100 mg/ml. The concentration-dependent absorption of protein molecules was determined by treating the solution as a two-component model first; then, based on protein absorptivity, the extent of the hydration shell is estimated. Protein molecules are shown to possess a concentration-dependent absorptivity in water solutions. Absorption curves of all three proteins sharply peak towards a dilution-limit that is attributed to the enhanced flexibility of protein and amino acid side chains. An alternative approach to the determination of hydration shell thickness is thereby suggested, based on protein absorptivity. The proposed approach is independent of the absorption of the hydration shell. The derived estimate of hydration shell thickness for each protein supports previous findings that protein-water interaction dynamics extends beyond 2-3 water solvation-layers as predicted by molecular dynamics simulations and other techniques such as NMR, X-ray scattering, and neutron scattering. According to our estimations, the radius of the dynamic hydration shell is 16, 19, and 25 Å, respectively, for lysozyme, myoglobin, and BSA proteins and correlates with the dipole moment of the protein. It is also seen that THz radiation can serve as an initial estimate of the protein hydrophobicity.

  11. Dynamics of Protein Expression Reveals Primary Targets and Secondary Messengers of Estrogen Receptor Alpha Signaling in MCF-7 Breast Cancer Cells.

    Science.gov (United States)

    Drabovich, Andrei P; Pavlou, Maria P; Schiza, Christina; Diamandis, Eleftherios P

    2016-06-01

    Estrogen receptor alpha (ERα)-mediated proliferation of breast cancer cells is facilitated through expression of multiple primary target genes, products of which induce a secondary response to stimulation. To differentiate between the primary and secondary target proteins of ERα signaling, we measured dynamics of protein expression induced by 17β-estradiol in MCF-7 breast cancer cells. Measurement of the global proteomic effects of estradiol by stable isotope labeling by amino acids in cell culture (SILAC) resulted in identification of 103 estrogen-regulated proteins, with only 40 of the corresponding genes having estrogen response elements. Selected reaction monitoring (SRM) assays were used to validate the differential expression of 19 proteins and measure the dynamics of their expression within 72 h after estradiol stimulation, and in the absence or presence of 4-hydroxytamoxifen, to confirm ERα-mediated signaling. Dynamics of protein expression unambiguously revealed early and delayed response proteins and well correlated with presence or absence of estrogen response elements in the corresponding genes. Finally, we quantified dynamics of protein expression in a rarely studied network of transcription factors with a negative feedback loop (ERα-EGR3-NAB2). Because NAB2 protein is a repressor of EGR3-induced transcription, siRNA-mediated silencing of NAB2 resulted in the enhanced expression of the EGR3-induced protein ITGA2. To conclude, we provided a high-quality proteomic resource to supplement genomic and transcriptomic studies of ERα signaling. PMID:27067054

  12. Comparative Proteomic Profiling of Ehrlichia ruminantium Pathogenic Strain and Its High-Passaged Attenuated Strain Reveals Virulence and Attenuation-Associated Proteins.

    Directory of Open Access Journals (Sweden)

    Isabel Marcelino

    Full Text Available The obligate intracellular bacterium Ehrlichia ruminantium (ER causes heartwater, a fatal tick-borne disease in livestock. In the field, ER strains present different levels of virulence, limiting vaccine efficacy, for which the molecular basis remains unknown. Moreover, there are no genetic tools currently available for ER manipulation, thus limiting the knowledge of the genes/proteins that are essential for ER pathogenesis and biology. As such, to identify proteins and/or mechanisms involved in ER virulence, we performed the first exhaustive comparative proteomic analysis between a virulent strain (ERGvir and its high-passaged attenuated strain (ERGatt. Despite their different behaviors in vivo and in vitro, our results from 1DE-nanoLC-MS/MS showed that ERGvir and ERGatt share 80% of their proteins; this core proteome includes chaperones, proteins involved in metabolism, protein-DNA-RNA biosynthesis and processing, and bacterial effectors. Conventional 2DE revealed that 85% of the identified proteins are proteoforms, suggesting that post-translational modifications (namely glycosylation are important in ER biology. Strain-specific proteins were also identified: while ERGatt has an increased number and overexpression of proteins involved in cell division, metabolism, transport and protein processing, ERGvir shows an overexpression of proteins and proteoforms (DIGE experiments involved in pathogenesis such as Lpd, AnkA, VirB9 and B10, providing molecular evidence for its increased virulence in vivo and in vitro. Overall, our work reveals that ERGvir and ERGatt proteomes are streamlined to fulfill their biological function (maximum virulence for ERGvir and replicative capacity for ERGatt, and we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.

  13. Phage-induced expression of CRISPR-associated proteins is revealed by shotgun proteomics in Streptococcus thermophilus.

    Directory of Open Access Journals (Sweden)

    Jacque C Young

    Full Text Available The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.

  14. Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis

    OpenAIRE

    Yu, Tao; Tao, Yonghui; Yang, Meiqiang; Chen, Peng; Gao, XiaoBo; Zhang, Yanbo; Zhang,Tao; Chen, Zi; Hou, Jian; Zhang, Yan; Ruan, Kangcheng; Wang, Hongyan; Hu, Ronggui

    2014-01-01

    Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by using bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be a...

  15. Phage-Induced Expression of CRISPR-Associated Proteins is Revealed by Shotgun Proteomics in Streptococcus thermophilus

    Energy Technology Data Exchange (ETDEWEB)

    Young, Jacque C [ORNL; Dill, Brian [ORNL; Pan, Chongle [ORNL; Hettich, Robert {Bob} L [ORNL; Banfield, Jillian F. [University of California, Berkeley; Shah, Manesh B [ORNL; Fremaux, Christophe [Danisco France SAS; Horvath, Philippe [Danisco France SAS; Barrangou, Rodolphe [Danisco USA; Verberkmoes, Nathan C [ORNL

    2012-01-01

    The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response: infection of S. thermophilus DGCC7710 with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectroscopy. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance during peak infection, including the Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.

  16. Label-free LC-MS/MS Analysis with Automated Phosphopeptide Enrichment Reveals Dynamic Human Milk Protein Phosphorylation during Lactation

    OpenAIRE

    Froehlich, John W.; Chu, Caroline S.; Tang, Ning; Waddell, Keith; Grimm, Rudolf; Lebrilla, Carlito B.

    2010-01-01

    Protein phosphorylation is a critical post-translational modification that affects cell-cell signaling and protein function. However, quantifying the relative, site-specific changes of phosphorylation occupancies remain a major issue. An on-line enrichment of phosphopeptides using titanium dioxide incorporated in a microchip liquid chromatography device was used to analyze trypsin-digested human milk proteins with mass spectrometry. The method was validated with standards, and utilized to det...

  17. Molecular dynamics simulations reveal that AEDANS is an inert fluorescent probe for the study of membrane proteins

    OpenAIRE

    2009-01-01

    Computer simulations were carried out of a number of AEDANS-labeled single cysteine mutants of a small reference membrane protein, M13 major coat protein, covering 60% of its primary sequence. M13 major coat protein is a single membrane-spanning, α-helical membrane protein with a relatively large water-exposed region in the N-terminus. In 10-ns molecular dynamics simulations, we analyze the behavior of the AEDANS label and the native tryptophan, which were used as acceptor and donor in previo...

  18. The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission

    International Nuclear Information System (INIS)

    The crystal structure of a monomeric mutant of Azami-Green (mAG) from G. fascicularis was determined at 2.2 Å resolution. Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green-emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino-acid sequences. mAG is more similar in its amino-acid sequence to four fluorescent proteins: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green-emitting fluorescent protein) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 Å crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants

  19. The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats

    DEFF Research Database (Denmark)

    Sarin, C T; Tack, B F; Kristensen, Torsten; Glenney Jr., J R; Hunter, T

    1986-01-01

    We have isolated and sequenced a full-length cDNA clone for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain). This sequence predicts a 339 amino acid (Mr 38,493) protein containing an N-terminal region of 20 amino acids, known to interact with a 10 kd protein (light chain), and...... A2 inhibitor lipocortin I were found to be 50% identical in sequence over the C-terminal 300 residues. The function of p36 and its relation to other proteins are discussed....

  20. Comparative genomics and experimental evolution of Escherichia coli BL21(DE3) strains reveal the landscape of toxicity escape from membrane protein overproduction.

    Science.gov (United States)

    Kwon, Soon-Kyeong; Kim, Seong Keun; Lee, Dae-Hee; Kim, Jihyun F

    2015-01-01

    Achieving sufficient yields of proteins in their functional form represents the first bottleneck in contemporary bioscience and biotechnology. To accomplish successful overexpression of membrane proteins in a workhorse organism such as E. coli, defined and rational optimization strategies based on an understanding of the genetic background of the toxicity-escape mechanism are desirable. To this end, we sequenced the genomes of E. coli C41(DE3) and its derivative C43(DE3), which were developed for membrane protein production. Comparative analysis of their genomes with those of their ancestral strain E. coli BL21(DE3) revealed various genetic changes in both strains. A series of E. coli variants that are able to tolerate transformation with or overexpression of membrane proteins were generated by in vitro evolution. Targeted sequencing of the evolved strains revealed the mutational hotspots among the acquired genetic changes. By these combinatorial approaches, we found non-synonymous changes in the lac repressor gene of the lac operon as well as nucleotide substitutions in the lacUV5 promoter of the DE3 region, by which the toxic effect to the host caused by overexpression of membrane proteins could be relieved. A mutation in lacI was demonstrated to be crucial for conferring tolerance to membrane protein overexpression. PMID:26531007

  1. Quantification of uncoupling protein 2 reveals its main expression in immune cells and selective up-regulation during T-cell proliferation.

    Directory of Open Access Journals (Sweden)

    Anne Rupprecht

    Full Text Available Uncoupling protein 2 (UCP2 is an inner mitochondrial membrane protein. Although the protein was discovered in 1997, its function and even its tissue distribution are still under debate. Here we present a quantitative analysis of mRNA and protein expression in various mice tissues, revealing that UCP2 is mainly expressed in organs and cells associated with the immune system. Although the UCP2 gene is present in the brain, as demonstrated using quantitative RT-PCR, the protein was not detectable in neurons under physiological conditions. Instead, we could detect UCP2 in microglia, which act in the immune defense of the central nervous system. In lymphocytes, activation led to a ten-fold increase of UCP2 protein expression simultaneously to the increase in levels of other mitochondrial proteins, whereas lymphocyte re-stimulation resulted in the selective increase of UCP2. The highest detected level of UCP2 expression in stimulated T-cells (0.54 ng/(µg total cellular protein was approximately 200 times lower than the level of UCP1 in brown adipose tissue from room temperature acclimated mice. Both the UCP2 expression pattern and the time course of up-regulation in stimulated T-cells imply UCP2's involvement in the immune response, probably by controlling the metabolism during cell proliferation.

  2. Phloem Proteomics Reveals New Lipid-Binding Proteins with a Putative Role in Lipid-Mediated Signaling.

    Science.gov (United States)

    Barbaglia, Allison M; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-01-01

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three

  3. Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

    Directory of Open Access Journals (Sweden)

    Allison Marie Barbaglia

    2016-04-01

    Full Text Available Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho- lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012. Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I a putative GDSL-motif lipase (II a PIG-P-like protein, with a possible receptor-like function; (III and PLAFP (phloem lipid-associated family protein, a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH, which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while

  4. The unique architecture and function of cellulose-interacting proteins in oomycetes revealed by genomic and structural analyses

    Directory of Open Access Journals (Sweden)

    Larroque Mathieu

    2012-11-01

    Full Text Available Abstract Background Oomycetes are fungal-like microorganisms evolutionary distinct from true fungi, belonging to the Stramenopile lineage and comprising major plant pathogens. Both oomycetes and fungi express proteins able to interact with cellulose, a major component of plant and oomycete cell walls, through the presence of carbohydrate-binding module belonging to the family 1 (CBM1. Fungal CBM1-containing proteins were implicated in cellulose degradation whereas in oomycetes, the Cellulose Binding Elicitor Lectin (CBEL, a well-characterized CBM1-protein from Phytophthora parasitica, was implicated in cell wall integrity, adhesion to cellulosic substrates and induction of plant immunity. Results To extend our knowledge on CBM1-containing proteins in oomycetes, we have conducted a comprehensive analysis on 60 fungi and 7 oomycetes genomes leading to the identification of 518 CBM1-containing proteins. In plant-interacting microorganisms, the larger number of CBM1-protein coding genes is expressed by necrotroph and hemibiotrophic pathogens, whereas a strong reduction of these genes is observed in symbionts and biotrophs. In fungi, more than 70% of CBM1-containing proteins correspond to enzymatic proteins in which CBM1 is associated with a catalytic unit involved in cellulose degradation. In oomycetes more than 90% of proteins are similar to CBEL in which CBM1 is associated with a non-catalytic PAN/Apple domain, known to interact with specific carbohydrates or proteins. Distinct Stramenopile genomes like diatoms and brown algae are devoid of CBM1 coding genes. A CBM1-PAN/Apple association 3D structural modeling was built allowing the identification of amino acid residues interacting with cellulose and suggesting the putative interaction of the PAN/Apple domain with another type of glucan. By Surface Plasmon Resonance experiments, we showed that CBEL binds to glycoproteins through galactose or N-acetyl-galactosamine motifs. Conclusions This study

  5. Molecular phylogeny of the kelch-repeat superfamily reveals an expansion of BTB/kelch proteins in animals

    Directory of Open Access Journals (Sweden)

    Adams Josephine C

    2003-09-01

    Full Text Available Abstract Background The kelch motif is an ancient and evolutionarily-widespread sequence motif of 44–56 amino acids in length. It occurs as five to seven repeats that form a β-propeller tertiary structure. Over 28 kelch-repeat proteins have been sequenced and functionally characterised from diverse organisms spanning from viruses, plants and fungi to mammals and it is evident from expressed sequence tag, domain and genome databases that many additional hypothetical proteins contain kelch-repeats. In general, kelch-repeat β-propellers are involved in protein-protein interactions, however the modest sequence identity between kelch motifs, the diversity of domain architectures, and the partial information on this protein family in any single species, all present difficulties to developing a coherent view of the kelch-repeat domain and the kelch-repeat protein superfamily. To understand the complexity of this superfamily of proteins, we have analysed by bioinformatics the complement of kelch-repeat proteins encoded in the human genome and have made comparisons to the kelch-repeat proteins encoded in other sequenced genomes. Results We identified 71 kelch-repeat proteins encoded in the human genome, whereas 5 or 8 members were identified in yeasts and around 18 in C. elegans, D. melanogaster and A. gambiae. Multiple domain architectures were identified in each organism, including previously unrecognised forms. The vast majority of kelch-repeat domains are predicted to form six-bladed β-propellers. The most prevalent domain architecture in the metazoan animal genomes studied was the BTB/kelch domain organisation and we uncovered 3 subgroups of human BTB/kelch proteins. Sequence analysis of the kelch-repeat domains of the most robustly-related subgroups identified differences in β-propeller organisation that could provide direction for experimental study of protein-binding characteristics. Conclusion The kelch-repeat superfamily constitutes a

  6. Electrophoretic behavior of DNA-methyl-CpG-binding domain protein complexes revealed by capillary electrophoreses laser-induced fluorescence.

    Science.gov (United States)

    Zhong, Shangwei; Zou, Dandan; Zhao, Bailin; Zhang, Dapeng; Li, Xiangjun; Wang, Hailin

    2015-12-01

    The free solution electrophoretic behavior of DNA-protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA-protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA-methyl-CpG-binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA-protein complexes. This method is based on the effective separation of free DNA and DNA-protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide-coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA-MBD2b complexes, we found that the values of (μ0 /μ)-1 were directly proportional to the charge-to-mass ratios of formed complexes, where the μ0 and μ are the mobility of free DNA probe and DNA-protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge-to-mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA-binding properties of the purified MBD2b protein and help to analyze other DNA-protein complexes. Additionally, this study enhances the understanding of the influence of the charge-to-mass ratios of formed DNA-protein complexes on their separation and electrophoretic behaviors. PMID:26377303

  7. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions

    OpenAIRE

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G.; Módos, Károly; Kardos, József; Liliom, Károly

    2010-01-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic,...

  8. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses.

    Science.gov (United States)

    Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

    2015-01-01

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system. PMID:26322064

  9. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    KAUST Repository

    Ali, Shawkat

    2015-08-11

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system.

  10. Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

    Science.gov (United States)

    Luo, Ting; Krüger, Thomas; Knüpfer, Uwe; Kasper, Lydia; Wielsch, Natalie; Hube, Bernhard; Kortgen, Andreas; Bauer, Michael; Giamarellos-Bourboulis, Evangelos J; Dimopoulos, George; Brakhage, Axel A; Kniemeyer, Olaf

    2016-08-01

    During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera. PMID:27386892

  11. Amyloid domains in the cell nucleus controlled by nucleoskeletal protein lamin B1 reveal a new pathway of mercury neurotoxicity

    Directory of Open Access Journals (Sweden)

    Florian Arnhold

    2015-02-01

    Full Text Available Mercury (Hg is a bioaccumulating trace metal that globally circulates the atmosphere and waters in its elemental, inorganic and organic chemical forms. While Hg represents a notorious neurotoxicant, the underlying cellular pathways are insufficiently understood. We identify amyloid protein aggregation in the cell nucleus as a novel pathway of Hg-bio-interactions. By mass spectrometry of purified protein aggregates, a subset of spliceosomal components and nucleoskeletal protein lamin B1 were detected as constituent parts of an Hg-induced nuclear aggregome network. The aggregome network was located by confocal imaging of amyloid-specific antibodies and dyes to amyloid cores within splicing-speckles that additionally recruit components of the ubiquitin-proteasome system. Hg significantly enhances global proteasomal activity in the nucleus, suggesting that formation of amyloid speckles plays a role in maintenance of protein homeostasis. RNAi knock down showed that lamin B1 for its part regulates amyloid speckle formation and thus likewise participates in nuclear protein homeostasis. As the Hg-induced cascade of interactions between the nucleoskeleton and protein homeostasis reduces neuronal signalling, amyloid fibrillation in the cell nucleus is introduced as a feature of Hg-neurotoxicity that opens new avenues of future research. Similar to protein aggregation events in the cytoplasm that are controlled by the cytoskeleton, amyloid fibrillation of nuclear proteins may be driven by the nucleoskeleton.

  12. Multi-omic profiling of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael;

    2015-01-01

    Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied t...

  13. Quantitative proteomic analysis of wheat grain proteins reveals differential effects of silencing of omega-5 gliadin genes in transgenic lines

    Science.gov (United States)

    Novel wheat lines with altered flour compositions can be used to decipher the roles of specific gluten proteins in flour quality. Grain proteins from transgenic wheat lines in which genes encoding the omega-5 gliadins were silenced by RNA interference (RNAi) were analyzed in detail by quantitative 2...

  14. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    Science.gov (United States)

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J; Molina, María

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

  15. A Protein Data Bank survey reveals shortening of intermolecular hydrogen bonds in ligand-protein complexes when a halogenated ligand is an H-bond donor.

    Directory of Open Access Journals (Sweden)

    Jarosław Poznański

    Full Text Available Halogen bonding in ligand-protein complexes is currently widely exploited, e.g. in drug design or supramolecular chemistry. But little attention has been directed to other effects that may result from replacement of a hydrogen by a strongly electronegative halogen. Analysis of almost 30000 hydrogen bonds between protein and ligand demonstrates that the length of a hydrogen bond depends on the type of donor-acceptor pair. Interestingly, lengths of hydrogen bonds between a protein and a halogenated ligand are visibly shorter than those estimated for the same family of proteins in complexes with non-halogenated ligands. Taking into account the effect of halogenation on hydrogen bonding is thus important when evaluating structural and/or energetic parameters of ligand-protein complexes. All these observations are consistent with the concept that halogenation increases the acidity of the proximal amino/imino/hydroxyl groups and thus makes them better, i.e. stronger, H-bond donors.

  16. Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.

    Directory of Open Access Journals (Sweden)

    Reshu Saxena

    Full Text Available Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1 through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01 in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1, VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61 and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein

  17. In-Depth N-Glycosylation Reveals Species-Specific Modifications and Functions of the Royal Jelly Protein from Western (Apis mellifera) and Eastern Honeybees (Apis cerana).

    Science.gov (United States)

    Feng, Mao; Fang, Yu; Han, Bin; Xu, Xiang; Fan, Pei; Hao, Yue; Qi, Yuping; Hu, Han; Huo, Xinmei; Meng, Lifeng; Wu, Bin; Li, Jianke

    2015-12-01

    Royal jelly (RJ), secreted by honeybee workers, plays diverse roles as nutrients and defense agents for honeybee biology and human health. Despite being reported to be glycoproteins, the glycosylation characterization and functionality of RJ proteins in different honeybee species are largely unknown. An in-depth N-glycoproteome analysis and functional assay of RJ produced by Apis mellifera lingustica (Aml) and Apis cerana cerana (Acc) were conducted. RJ produced by Aml yielded 80 nonredundant N-glycoproteins carrying 190 glycosites, of which 23 novel proteins harboring 35 glycosites were identified. For Acc, all 43 proteins glycosylated at 138 glycosites were reported for the first time. Proteins with distinct N-glycoproteomic characteristics in terms of glycoprotein species, number of N-glycosylated sites, glycosylation motif, abundance level of glycoproteins, and N-glycosites were observed in this two RJ samples. The fact that the low inhibitory efficiency of N-glycosylated major royal jelly protein 2 (MRJP2) against Paenibacillus larvae (P. larvae) and the absence of antibacterial related glycosylated apidaecin, hymenoptaecin, and peritrophic matrix in the Aml RJ compared to Acc reveal the mechanism for why the Aml larvae are susceptible to P. larvae, the causative agent of a fatal brood disease (American foulbrood, AFB). The observed antihypertension activity of N-glycosylated MRJP1 in two RJ samples and a stronger activity found in Acc than in Aml reveal that specific RJ protein and modification are potentially useful for the treatment of hypertensive disease for humans. Our data gain novel understanding that the western and eastern bees have evolved species-specific strategies of glycosylation to fine-tune protein activity for optimizing molecular function as nutrients and immune agents for the good of honeybee and influence on the health promoting activity for human as well. This serves as a valuable resource for the targeted probing of the biological

  18. The proteomic investigation reveals interaction of mdig protein with the machinery of DNA double-strand break repair.

    Science.gov (United States)

    Wang, Wei; Lu, Yongju; Stemmer, Paul M; Zhang, Xiangmin; Bi, Yongyi; Yi, Zhengping; Chen, Fei

    2015-09-29

    To investigate how mineral dust-induced gene (mdig, also named as mina53, MINA, or NO52) promotes carcinogenesis through inducing active chromatin, we performed proteomics analyses for the interacting proteins that were co-immunoprecipitated by anti-mdig antibody from either the lung cancer cell line A549 cells or the human bronchial epithelial cell line BEAS-2B cells. On SDS-PAGE gels, three to five unique protein bands were consistently observed in the complexes pulled-down by mdig antibody, but not the control IgG. In addition to the mdig protein, several DNA repair or chromatin binding proteins, including XRCC5, XRCC6, RBBP4, CBX8, PRMT5, and TDRD, were identified in the complexes by the proteomics analyses using both Orbitrap Fusion and Orbitrap XL nanoESI-MS/MS in four independent experiments. The interaction of mdig with some of these proteins was further validated by co-immunoprecipitation using antibodies against mdig and its partner proteins, respectively. These data, thus, provide evidence suggesting that mdig accomplishes its functions on chromatin, DNA repair and cell growth through interacting with the partner proteins. PMID:26293673

  19. Phosphoproteomic analysis reveals major default phosphorylation sites outside long intrinsically disordered regions of Arabidopsis plasma membrane proteins

    Directory of Open Access Journals (Sweden)

    Nespoulous Claude

    2012-10-01

    Full Text Available Abstract Background Genome-wide statistics established that long intrinsically disordered regions (over 30 residues are predicted in a large part of proteins in all eukaryotes, with a higher ratio in trans-membrane proteins. At functional level, such unstructured and flexible regions were suggested for years to favour phosphorylation events. In plants, despite increasing evidence of the regulation of transport and signalling processes by phosphorylation events, only few data are available without specific information regarding plasma membrane proteins, especially at proteome scale. Results Using a dedicated phosphoproteomic workflow, 75 novel and unambiguous phosphorylation sites were identified in Arabidopsis plasma membrane. Bioinformatics analysis showed that this new dataset concerned mostly integral proteins involved in key functions of the plasma membrane (such as transport and signal transduction, including protein phosphorylation. It thus expanded by 15% the directory of phosphosites previously characterized in signalling and transport proteins. Unexpectedly, 66% of phosphorylation sites were predicted to be located outside long intrinsically disordered regions. This result was further corroborated by analysis of publicly available data for the plasma membrane. Conclusions The new phosphoproteomics data presented here, with published datasets and functional annotation, suggest a previously unexpected topology of phosphorylation in the plant plasma membrane proteins. The significance of these new insights into the so far overlooked properties of the plant plasma membrane phosphoproteome and the long disordered regions is discussed.

  20. Comparison of the aggregation of homologous β2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation.

    Science.gov (United States)

    Pashley, Clare L; Hewitt, Eric W; Radford, Sheena E

    2016-02-13

    The mouse and human β2-microglobulin protein orthologs are 70 % identical in sequence and share 88 % sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human β2m (hβ2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH2.5) in the absence of NaCl, mouse β2m (mβ2m) requires the addition of 0.3M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hβ2m and mβ2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation. PMID:26780548

  1. Comparative proteomics reveals key proteins recruited at the nucleoid of Deinococcus after irradiation-induced DNA damage

    International Nuclear Information System (INIS)

    The nucleoids of radiation-resistant Deinococcus species show a high degree of compaction maintained after ionizing irradiation. We identified proteins recruited after irradiation in nucleoids of Deinococcus radiodurans and Deinococcus deserti by means of comparative proteomics. Proteins in nucleoid-enriched fractions from unirradiated and irradiated Deinococcus were identified and semi quantified by shotgun proteomics. The ssDNA-binding protein SSB, DNA gyrase subunits GyrA and GyrB, DNA topoisomerase I, RecA recombinase, UvrA excinuclease, RecQ helicase, DdrA, DdrB, and DdrD proteins were found in significantly higher amounts in irradiated nucleoids of both Deinococcus species. We observed, by immunofluorescence microscopy, the subcellular localization of these proteins in D. radiodurans, showing for the first time the recruitment of the DdrD protein into the D. radiodurans nucleoid. We specifically followed the kinetics of recruitment of RecA, DdrA, and DdrD to the nucleoid after irradiation. Remarkably, RecA proteins formed irregular filament-like structures 1 h after irradiation, before being redistributed throughout the cells by 3 h post-irradiation. Comparable dynamics of DdrD localization were observed, suggesting a possible functional interaction between RecA and DdrD. Several proteins involved in nucleotide synthesis were also seen in higher quantities in the nucleoids of irradiated cells, indicative of the existence of a mechanism for orchestrating the presence of proteins involved in DNA metabolism in nucleoids in response to massive DNA damage. All MS data have been deposited in the ProteomeXchange with identifier PXD00196. (authors)

  2. Molecular contacts for chlorosome envelope proteins revealed by cross-linking studies with chlorosomes from Chlorobium tepidum

    DEFF Research Database (Denmark)

    Li, Hui; Frigaard, Niels-Ulrik; Bryant, Donald A

    2006-01-01

    replaced with glutaraldehyde. Specific interactions between chlorosome proteins in cross-linked products were identified by immunoblotting with polyclonal antibodies raised against recombinant chlorosome proteins. We confirmed these interactions by demonstrating that these products were missing in...... type and mutants lacking a single chlorosome protein were cross-linked with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and analyzed by gel electrophoresis. Similar cross-linking products were observed when the time and temperature were varied or when EDC was...

  3. Antibodies to the core proteins of Nairobi sheep disease virus/Ganjam virus reveal details of the distribution of the proteins in infected cells and tissues.

    Science.gov (United States)

    Lasecka, Lidia; Bin-Tarif, Abdelghani; Bridgen, Anne; Juleff, Nicholas; Waters, Ryan A; Baron, Michael D

    2015-01-01

    Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages. PMID:25905707

  4. Proteomic and biochemical analyses reveal the activation of unfolded protein response, ERK-1/2 and ribosomal protein S6 signaling in experimental autoimmune myocarditis rat model

    Directory of Open Access Journals (Sweden)

    Kim Chan

    2011-10-01

    Full Text Available Abstract Background To investigate the molecular and cellular pathogenesis underlying myocarditis, we used an experimental autoimmune myocarditis (EAM-induced heart failure rat model that represents T cell mediated postinflammatory heart disorders. Results By performing unbiased 2-dimensional electrophoresis of protein extracts from control rat heart tissues and EAM rat heart tissues, followed by nano-HPLC-ESI-QIT-MS, 67 proteins were identified from 71 spots that exhibited significantly altered expression levels. The majority of up-regulated proteins were confidently associated with unfolded protein responses (UPR, while the majority of down-regulated proteins were involved with the generation of precursor metabolites and energy metabolism in mitochondria. Although there was no difference in AKT signaling between EAM rat heart tissues and control rat heart tissues, the amounts and activities of extracellular signal-regulated kinase (ERK-1/2 and ribosomal protein S6 (rpS6 were significantly increased. By comparing our data with the previously reported myocardial proteome of the Coxsackie viruses of group B (CVB-mediated myocarditis model, we found that UPR-related proteins were commonly up-regulated in two murine myocarditis models. Even though only two out of 29 down-regulated proteins in EAM rat heart tissues were also dysregulated in CVB-infected rat heart tissues, other proteins known to be involved with the generation of precursor metabolites and energy metabolism in mitochondria were also dysregulated in CVB-mediated myocarditis rat heart tissues, suggesting that impairment of mitochondrial functions may be a common underlying mechanism of the two murine myocarditis models. Conclusions UPR, ERK-1/2 and S6RP signaling were activated in both EAM- and CVB-induced myocarditis murine models. Thus, the conserved components of signaling pathways in two murine models of acute myocarditis could be targets for developing new therapeutic drugs or

  5. Identification of Novel Proteins Co-Purifying with Cockayne Syndrome Group B (CSB Reveals Potential Roles for CSB in RNA Metabolism and Chromatin Dynamics.

    Directory of Open Access Journals (Sweden)

    Serena Nicolai

    Full Text Available The CSB protein, a member of the SWI/SNF ATP dependent chromatin remodeling family of proteins, plays a role in a sub-pathway of nucleotide excision repair (NER known as transcription coupled repair (TCR. CSB is frequently mutated in Cockayne syndrome group B, a segmental progeroid human autosomal recessive disease characterized by growth failure and degeneration of multiple organs. Though initially classified as a DNA repair protein, recent studies have demonstrated that the loss of CSB results in pleiotropic effects. Identification of novel proteins belonging to the CSB interactome may be useful not only for predicting the molecular basis for diverse pathological symptoms of CS-B patients but also for unraveling the functions of CSB in addition to its authentic role in DNA repair. In this study, we performed tandem affinity purification (TAP technology coupled with mass spectrometry and co-immunoprecipitation studies to identify and characterize the proteins that potentially interact with CSB-TAP. Our approach revealed 33 proteins that were not previously known to interact with CSB. These newly identified proteins indicate potential roles for CSB in RNA metabolism involving repression and activation of transcription process and in the maintenance of chromatin dynamics and integrity.

  6. Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Leon, Ileana R; Bak, Steffen;

    2011-01-01

    Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skel...

  7. Interactome Analysis of the NS1 Protein Encoded by Influenza A H1N1 Virus Reveals a Positive Regulatory Role of Host Protein PRP19 in Viral Replication.

    Science.gov (United States)

    Kuo, Rei-Lin; Li, Zong-Hua; Li, Li-Hsin; Lee, Kuo-Ming; Tam, Ee-Hong; Liu, Helene M; Liu, Hao-Ping; Shih, Shin-Ru; Wu, Chih-Ching

    2016-05-01

    Influenza A virus, which can cause severe respiratory illnesses in infected individuals, is responsible for worldwide human pandemics. The NS1 protein encoded by this virus plays a crucial role in regulating the host antiviral response through various mechanisms. In addition, it has been reported that NS1 can modulate cellular pre-mRNA splicing events. To investigate the biological processes potentially affected by the NS1 protein in host cells, NS1-associated protein complexes in human cells were identified using coimmunoprecipitation combined with GeLC-MS/MS. By employing software to build biological process and protein-protein interaction networks, NS1-interacting cellular proteins were found to be related to RNA splicing/processing, cell cycle, and protein folding/targeting cellular processes. By monitoring spliced and unspliced RNAs of a reporter plasmid, we further validated that NS1 can interfere with cellular pre-mRNA splicing. One of the identified proteins, pre-mRNA-processing factor 19 (PRP19), was confirmed to interact with the NS1 protein in influenza A virus-infected cells. Importantly, depletion of PRP19 in host cells reduced replication of influenza A virus. In summary, the interactome of influenza A virus NS1 in host cells was comprehensively profiled, and our findings reveal a novel regulatory role for PRP19 in viral replication. PMID:27096427

  8. Electrophoretic analysis of the major outer membrane protein of Chlamydia psittaci reveals multimers which are recognized by protective monoclonal antibodies.

    OpenAIRE

    McCafferty, M C; Herring, A J; Andersen, A A; Jones, G. E.

    1995-01-01

    Purified major outer membrane protein, detergent solubilized and reduced with dithiothreitol but not heated, gave an apparent molecular weight in sodium dodecyl sulfate (SDS)-polyacrylamide gels almost three times that observed for the heat-denatured SDS-treated peptide. This is similar to the behavior of porin trimers from gram-negative bacteria. Two protective monoclonal antibodies showed strong binding to the proposed trimer but not to denatured, monomeric major outer membrane protein.

  9. In vivo expression of mRNA for the Ca++-binding protein SPARC (osteonectin) revealed by in situ hybridization

    OpenAIRE

    1987-01-01

    In situ hybridization is used to survey the tissue-specific and developmental expression of the cloned mouse gene Sparc, coding for a protein homologous to the bovine Ca++-binding protein, osteonectin. High levels of SPARC RNA are found in osteoblasts and odontoblasts. In addition, high grain counts are associated with a variety of other cell types in the embryo and newborn mouse, including parietal endoderm, deciduum, whisker follicles (connective tissue sheath), peripheral nerve trunk, skin...

  10. Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Feng; Waters, Katrina M.; Miller, John H.; Gritsenko, Marina A.; Zhao, Rui; Du, Xiuxia; Livesay, Eric A.; Purvine, Samuel O.; Monroe, Matthew E.; Wang, Yingchun; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2010-11-30

    Background: High doses of ionizing radiation result in biological damage, however the precise relationships between long term health effects, including cancer, and low dose exposures remain poorly understood and are currently extrapolated using high dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose dependent responses to radiation. Principle Findings: We have identified 6845 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts one hour post-exposure. Dual statistical analyses based on spectral counts and peak intensities identified 287 phosphopeptides (from 231 proteins) and 244 phosphopeptides (from 182 proteins) that varied significantly following exposure to 2 and 50 cGy respectively. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatics analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role of MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. Conlcusions: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provides a basis for the systems level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at

  11. Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging

    OpenAIRE

    Hua, Wei; Sheff, David; Toomre, Derek; Mellman, Ira

    2006-01-01

    Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin–Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi n...

  12. Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

    OpenAIRE

    Verdoliva, Vincenzo; Senatore, Cinzia; Polci, Maria Letizia; Rossi, Stefania; Cordella, Martina; Carlucci, Giuseppe; Marchetti, Paolo; Antonini-Cappellini, Giancarlo; Facchiano, Antonio; D'Arcangelo, Daniela; Facchiano, Francesco

    2013-01-01

    Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different ...

  13. Beta-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors.

    OpenAIRE

    Azzi, Mounia; Pascale G. Charest; Angers, Stéphane; Rousseau, Guy; Kohout, Trudy; Bouvier, Michel; Piñeyro, Graciela

    2003-01-01

    It is becoming increasingly clear that signaling via G protein-coupled receptors is a diverse phenomenon involving receptor interaction with a variety of signaling partners. Despite this diversity, receptor ligands are commonly classified only according to their ability to modify G protein-dependent signaling. Here we show that beta2AR ligands like ICI118551 and propranolol, which are inverse agonists for Gs-stimulated adenylyl cyclase, induce partial agonist responses for the mitogen-activat...

  14. Isolation and Characterization of the Prochlorococcus Carboxysome Reveal the Presence of the Novel Shell Protein CsoS1D

    OpenAIRE

    Roberts, Evan W.; Cai, Fei; Cheryl A Kerfeld; Cannon, Gordon C.; Heinhorst, Sabine

    2012-01-01

    Cyanobacteria, including members of the genus Prochlorococcus, contain icosahedral protein microcompartments known as carboxysomes that encapsulate multiple copies of the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in a thin protein shell that enhances the catalytic performance of the enzyme in part through the action of a shell-associated carbonic anhydrase. However, the exact mechanism by which compartmentation provides a catalytic advantage to the enzyme is ...

  15. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

    Science.gov (United States)

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

  16. Proteomic Profiling of the Dystrophin-Deficient MDX Heart Reveals Drastically Altered Levels of Key Metabolic and Contractile Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Lewis

    2010-01-01

    Full Text Available Although Duchenne muscular dystrophy is primarily classified as a neuromuscular disease, cardiac complications play an important role in the course of this X-linked inherited disorder. The pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood. We therefore carried out a fluorescence difference in-gel electrophoretic analysis of 9-month-old dystrophin-deficient versus age-matched normal heart, using the established MDX mouse model of muscular dystrophy-related cardiomyopathy. Out of 2,509 detectable protein spots, 79 2D-spots showed a drastic differential expression pattern, with the concentration of 3 proteins being increased, including nucleoside diphosphate kinase and lamin-A/C, and of 26 protein species being decreased, including ATP synthase, fatty acid binding-protein, isocitrate dehydrogenase, NADH dehydrogenase, porin, peroxiredoxin, adenylate kinase, tropomyosin, actin, and myosin light chains. Hence, the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the MDX heart, affecting especially energy metabolism and contractile proteins.

  17. Central Nervous System Proteomics in Animal Model of Multiple Sclerosis Revealed Down-Regulation of Mithochondrial Proteins

    Directory of Open Access Journals (Sweden)

    Abolhassan Shahzadeh Fazeli

    2009-01-01

    Full Text Available Objective: Detection of central nervous system (CNS molecular defects in an animal modelof multiple sclerosis.Materials and Methods: Experimental autoimmune encephalomyelitis (EAE was inducedby a myelin oligodendrocyte glycoprotein. Protein expression profiles in the central nervoussystem between healthy clinical scores 1 and 3 of EAE were studied using a two dimensionalelectrophoresis based proteomics approach coupled with MALDI TOF/TOF massspectrometry.Results: We identified 8 mitochondrial proteins that were differentially expressed in CNS, allof them down-regulated in scores 1 and/or 3. Of these, 5 proteins belong to the mitochondrialrespiratory chain including: NADH dehydrogenase (ubiquinone Fe-S protein 8, cytochromec oxidase Va, cytochrome c oxidase Vb, ATP5B, NADH dehydrogenase (ubiquinone flavoprotein2. We also observed down-regulation of three other mitochondrial proteins including:glutaredoxin 5, estradiol 17 beta-dehydrogenase 8 and isocitrate dehydrogenase.Conclusion: Down-regulation of mitochondrial proteins supported the hypothesis thathypoxia-like tissue injury in multiple sclerosis (MS lesions may be due to mitochondrialimpairment.

  18. Isolation and characterization of the Prochlorococcus carboxysome reveal the presence of the novel shell protein CsoS1D.

    Science.gov (United States)

    Roberts, Evan W; Cai, Fei; Kerfeld, Cheryl A; Cannon, Gordon C; Heinhorst, Sabine

    2012-02-01

    Cyanobacteria, including members of the genus Prochlorococcus, contain icosahedral protein microcompartments known as carboxysomes that encapsulate multiple copies of the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in a thin protein shell that enhances the catalytic performance of the enzyme in part through the action of a shell-associated carbonic anhydrase. However, the exact mechanism by which compartmentation provides a catalytic advantage to the enzyme is not known. Complicating the study of cyanobacterial carboxysomes has been the inability to obtain homogeneous carboxysome preparations. This study describes the first successful purification and characterization of carboxysomes from the marine cyanobacterium Prochlorococcus marinus MED4. Because the isolated P. marinus MED4 carboxysomes were free from contaminating membrane proteins, their protein complement could be assessed. In addition to the expected shell proteins, the CsoS1D protein that is not encoded by the canonical cso gene clusters of α-cyanobacteria was found to be a low-abundance shell component. This finding and supporting comparative genomic evidence have important implications for carboxysome composition, structure, and function. Our study indicates that carboxysome composition is probably more complex than was previously assumed based on the gene complements of the classical cso gene clusters. PMID:22155772

  19. Proteomic screening of glucose-responsive and glucose non-reponsive MIN-6 beta cells reveals differential expression of protein involved in protein folding, secretion and oxidative stress

    DEFF Research Database (Denmark)

    Dowling, P.; O´Driscoll, L.; O´Sullivan, F.;

    2006-01-01

    The glucose-sensitive insulin-secretion (GSIS) phenotype is relatively unstable in long-term culture of beta cells. The purpose of this study was to investigate relative changes in the proteome between glucose-responsive (low passage) and glucose non-responsive (high passage) murine MIN-6...... pancreatic beta cells. The 2D-DIGE and subsequent DeCyder analysis detected 3351 protein spots in the pH range of 4-7. Comparing MIN-6(H) to MIN-6(L) and using a threshold of 1.2-fold, the number of proteins with a decrease in expression level was 152 (4.5%), similar was 3140 (93.7%) and increased 59 (1...... protein 29 (ERp29); 78-kDa glucose-related protein, (GRP78); 94-kDa glucose-related protein (GRP94); protein disulphide isomerase; carbonyl reductase 3; peroxidoxin 4 and superoxide dismutase 1. These results suggest that non-GSIS MIN-6 cells do not have the same ability/capacity of glucose-responsive MIN...

  20. SAFER, an Analysis Method of Quantitative Proteomic Data, Reveals New Interactors of the C. elegans Autophagic Protein LGG-1.

    Science.gov (United States)

    Yi, Zhou; Manil-Ségalen, Marion; Sago, Laila; Glatigny, Annie; Redeker, Virginie; Legouis, Renaud; Mucchielli-Giorgi, Marie-Hélène

    2016-05-01

    Affinity purifications followed by mass spectrometric analysis are used to identify protein-protein interactions. Because quantitative proteomic data are noisy, it is necessary to develop statistical methods to eliminate false-positives and identify true partners. We present here a novel approach for filtering false interactors, named "SAFER" for mass Spectrometry data Analysis by Filtering of Experimental Replicates, which is based on the reproducibility of the replicates and the fold-change of the protein intensities between bait and control. To identify regulators or targets of autophagy, we characterized the interactors of LGG1, a ubiquitin-like protein involved in autophagosome formation in C. elegans. LGG-1 partners were purified by affinity, analyzed by nanoLC-MS/MS mass spectrometry, and quantified by a label-free proteomic approach based on the mass spectrometric signal intensity of peptide precursor ions. Because the selection of confident interactions depends on the method used for statistical analysis, we compared SAFER with several statistical tests and different scoring algorithms on this set of data. We show that SAFER recovers high-confidence interactors that have been ignored by the other methods and identified new candidates involved in the autophagy process. We further validated our method on a public data set and conclude that SAFER notably improves the identification of protein interactors. PMID:26999449

  1. RNA-Seq reveals 10 novel promising candidate genes affecting milk protein concentration in the Chinese Holstein population.

    Science.gov (United States)

    Li, Cong; Cai, Wentao; Zhou, Chenghao; Yin, Hongwei; Zhang, Ziqi; Loor, Juan J; Sun, Dongxiao; Zhang, Qin; Liu, Jianfeng; Zhang, Shengli

    2016-01-01

    Paired-end RNA sequencing (RNA-Seq) was used to explore the bovine transcriptome from the mammary tissue of 12 Chinese Holstein cows with 6 extremely high and 6 low phenotypic values for milk protein percentage. We defined the differentially expressed transcripts between the two comparison groups, extremely high and low milk protein percentage during the peak lactation (HP vs LP) and during the non-lactating period (HD vs LD), respectively. Within the differentially expressed genes (DEGs), we detected 157 at peak lactation and 497 in the non-lactating period with a highly significant correlation with milk protein concentration. Integrated interpretation of differential gene expression indicated that SERPINA1, CLU, CNTFR, ERBB2, NEDD4L, ANG, GALE, HSPA8, LPAR6 and CD14 are the most promising candidate genes affecting milk protein concentration. Similarly, LTF, FCGR3A, MEGF10, RRM2 and UBE2C are the most promising candidates that in the non-lactating period could help the mammary tissue prevent issues with inflammation and udder disorders. Putative genes will be valuable resources for designing better breeding strategies to optimize the content of milk protein and also to provide new insights into regulation of lactogenesis. PMID:27254118

  2. Systematic Mapping of Protein Mutational Space by Prolonged Drift Reveals the Deleterious Effects of Seemingly Neutral Mutations.

    Directory of Open Access Journals (Sweden)

    Liat Rockah-Shmuel

    2015-08-01

    Full Text Available Systematic mappings of the effects of protein mutations are becoming increasingly popular. Unexpectedly, these experiments often find that proteins are tolerant to most amino acid substitutions, including substitutions in positions that are highly conserved in nature. To obtain a more realistic distribution of the effects of protein mutations, we applied a laboratory drift comprising 17 rounds of random mutagenesis and selection of M.HaeIII, a DNA methyltransferase. During this drift, multiple mutations gradually accumulated. Deep sequencing of the drifted gene ensembles allowed determination of the relative effects of all possible single nucleotide mutations. Despite being averaged across many different genetic backgrounds, about 67% of all nonsynonymous, missense mutations were evidently deleterious, and an additional 16% were likely to be deleterious. In the early generations, the frequency of most deleterious mutations remained high. However, by the 17th generation, their frequency was consistently reduced, and those remaining were accepted alongside compensatory mutations. The tolerance to mutations measured in this laboratory drift correlated with sequence exchanges seen in M.HaeIII's natural orthologs. The biophysical constraints dictating purging in nature and in this laboratory drift also seemed to overlap. Our experiment therefore provides an improved method for measuring the effects of protein mutations that more closely replicates the natural evolutionary forces, and thereby a more realistic view of the mutational space of proteins.

  3. Atomic Force Microscope Spectroscopy Reveals a Hemifusion Intermediate during Soluble N-Ethylmaleimide-Sensitive Factor-Attachment Protein Receptors-Mediated Membrane Fusion

    OpenAIRE

    Abdulreda, Midhat H.; Bhalla, Akhil; Chapman, Edwin R.; Moy, Vincent T.

    2007-01-01

    This study investigated the effect of soluble N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptors (SNAREs) on the fusion of egg L-α-phosphatidylcholine bilayers using atomic force microscope (AFM) spectroscopy. AFM measurements of the fusion force under compression were acquired to reveal the energy landscape of the fusion process. A single main energy barrier governing the fusion process was identified in the absence and presence of SNAREs in the bilayers. Under compression...

  4. Antibody-based analysis reveals “filamentous vs. non-filamentous” and “cytoplasmic vs. nuclear” crosstalk of cytoskeletal proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kumeta, Masahiro, E-mail: kumeta@lif.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Kyoto 606-8501 (Japan); Hirai, Yuya; Yoshimura, Shige H. [Graduate School of Biostudies, Kyoto University, Kyoto 606-8501 (Japan); Horigome, Tsuneyoshi [Graduate School of Science and Technology, Niigata University, Niigata 950-2181 (Japan); Takeyasu, Kunio [Graduate School of Biostudies, Kyoto University, Kyoto 606-8501 (Japan)

    2013-12-10

    To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus. - Highlights: • A set of monoclonal antibodies were raised against nuclear scaffold proteins. • Helix-based cytoskeletal proteins were involved in nuclear scaffold. • Many cytoskeletal components shuttle into the nucleus in a CRM1-dependent manner. • Sets of antibodies distinguished distinct subcellular localization of a single isoform. • Nuclear keratin is soluble and does not form an obvious filamentous structure.

  5. Proteomic Dissection of Endosperm Starch Granule Associated Proteins Reveals a Network Coordinating Starch Biosynthesis and Amino Acid Metabolism and Glycolysis in Rice Endosperms

    Science.gov (United States)

    Yu, Huatao; Wang, Tai

    2016-01-01

    Starch biosynthesis and starch granule packaging in cereal endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central endosperm at 15 DAF and in the whole endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal endosperms. PMID:27252723

  6. Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis

    DEFF Research Database (Denmark)

    Holst, B; Hastrup, H; Raffetseder, U; Martini, L; Schwartz, T W

    2001-01-01

    either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P...... especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to......The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between...

  7. Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation.

    Directory of Open Access Journals (Sweden)

    Feng Yang

    Full Text Available BACKGROUND: High doses of ionizing radiation result in biological damage; however, the precise relationships between long-term health effects, including cancer, and low-dose exposures remain poorly understood and are currently extrapolated using high-dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose-dependent responses to radiation. PRINCIPAL FINDINGS: We have identified 7117 unique phosphopeptides (2566 phosphoproteins from control and irradiated (2 and 50 cGy primary human skin fibroblasts 1 h post-exposure. Semi-quantitative label-free analyses were performed to identify phosphopeptides that are apparently altered by radiation exposure. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation-responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatic analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role for MAP kinase and protein kinase A (PKA signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. CONCLUSIONS: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provide a basis for the systems-level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low-dose radiation exposure on human health.

  8. Comparison of the aggregation of homologous β2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation

    OpenAIRE

    Pashley, Clare L.; Hewitt, Eric W.; Sheena E. Radford

    2016-01-01

    The mouse and human β2-microglobulin protein orthologs are 70 % identical in sequence and share 88 % sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human β2m (hβ2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH 2.5) in the absence of NaCl, mouse β2m (mβ2m) requires the addition of 0.3 M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propens...

  9. Drosophila snRNP associated protein P11 which specifically binds to heat shock puff 93D reveals strong homology with hnRNP core protein A1.

    OpenAIRE

    Hovemann, Bernhard; Dessen, Eliana; Mechler, Hans; Mack, Elke

    1991-01-01

    We have isolated cDNAs coding for a ribonucleoprotein of Drosophila melanogaster that is distinguished by its nearly exclusive presence at only one of the several heat shock puffs in polytene chromosomes of third instar larvae. We determined the nucleotide sequence and deduced the corresponding amino acid sequence. Its coding capacity for a 39 kDa protein is consistent with the size of the protein detected by the monoclonal antibody P11 used for expression cloning. Our results show that the P...

  10. Analysis of the Sequence and Phenotype of Drosophila Sex combs reduced Alleles Reveals Potential Functions of Conserved Protein Motifs of the Sex combs reduced Protein

    OpenAIRE

    Sivanantharajah, Lovesha; Percival-Smith, Anthony

    2009-01-01

    The Drosophila Hox gene, Sex combs reduced (Scr), is required for patterning the larval and adult, labial and prothoracic segments. Fifteen Scr alleles were sequenced and the phenotypes analyzed in detail. Six null alleles were nonsense mutations (Scr2, Scr4, Scr11, Scr13, Scr13A, and Scr16) and one was an intragenic deletion (Scr17). Five hypomorphic alleles were missense mutations (Scr1, Scr3, Scr5, Scr6, and Scr8) and one was a small protein deletion (Scr15). Protein sequence changes were ...

  11. Novel circular single-stranded DNA viruses identified in marine invertebrates reveal high sequence diversity and consistent predicted intrinsic disorder patterns within putative structural proteins

    Directory of Open Access Journals (Sweden)

    Karyna eRosario

    2015-07-01

    Full Text Available Viral metagenomics has recently revealed the ubiquitous and diverse nature of single-stranded DNA (ssDNA viruses that encode a conserved replication initiator protein (Rep in the marine environment. Although eukaryotic circular Rep-encoding ssDNA (CRESS-DNA viruses were originally thought to only infect plants and vertebrates, recent studies have identified these viruses in a number of invertebrates. To further explore CRESS-DNA viruses in the marine environment, this study surveyed CRESS-DNA viruses in various marine invertebrate species. A total of 27 novel CRESS-DNA genomes, with Reps that share less than 60.1% identity with previously reported viruses, were recovered from 21 invertebrate species, mainly crustaceans. Phylogenetic analysis based on the Rep revealed a novel clade of CRESS-DNA viruses that included approximately one third of the marine invertebrate associated viruses identified here and whose members may represent a novel family. Investigation of putative capsid proteins (Cap encoded within the eukaryotic CRESS-DNA viral genomes from this study and those in GenBank demonstrated conserved patterns of predicted intrinsically disordered regions (IDRs, which can be used to complement similarity-based searches to identify divergent structural proteins within novel genomes. Overall, this study expands our knowledge of CRESS-DNA viruses associated with invertebrates and explores a new tool to evaluate divergent structural proteins encoded by these viruses.

  12. iTRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis.

    Science.gov (United States)

    Luczak, Magdalena; Formanowicz, Dorota; Marczak, Łukasz; Suszyńska-Zajczyk, Joanna; Pawliczak, Elżbieta; Wanic-Kossowska, Maria; Stobiecki, Maciej

    2016-01-01

    Patients with chronic kidney disease (CKD) have a considerably higher risk of death due to cardiovascular causes. Using an iTRAQ MS/MS approach, we investigated the alterations in plasma protein accumulation in patients with CKD and classical cardiovascular disease (CVD) without CKD. The proteomic analysis led to the identification of 130 differentially expressed proteins among CVD and CKD patients and healthy volunteers. Bioinformatics analysis revealed that 29 differentially expressed proteins were involved in lipid metabolism and atherosclerosis, 20 of which were apolipoproteins and constituents of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Although dyslipidemia is common in CKD patients, we found that significant changes in apolipoproteins were not strictly associated with changes in plasma lipid levels. A lack of correlation between apoB and LDL concentration and an inverse relationship of some proteins with the HDL level were revealed. An increased level of apolipoprotein AIV, adiponectin, or apolipoprotein C, despite their anti-atherogenic properties, was not associated with a decrease in cardiovascular event risk in CKD patients. The presence of the distinctive pattern of apolipoproteins demonstrated in this study may suggest that lipid abnormalities in CKD are characterized by more qualitative abnormalities and may be related to HDL function rather than HDL deficiency. PMID:27600335

  13. Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus.

    Science.gov (United States)

    Rey-Burusco, M Florencia; Ibáñez-Shimabukuro, Marina; Gabrielsen, Mads; Franchini, Gisela R; Roe, Andrew J; Griffiths, Kate; Zhan, Bin; Cooper, Alan; Kennedy, Malcolm W; Córsico, Betina; Smith, Brian O

    2015-11-01

    Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein-ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male. PMID:26318523

  14. Combined probes of X-ray scattering and optical spectroscopy reveal how global conformational change is temporally and spatially linked to local structural perturbation in photoactive yellow protein.

    Science.gov (United States)

    Kim, Tae Wu; Yang, Cheolhee; Kim, Youngmin; Kim, Jong Goo; Kim, Jeongho; Jung, Yang Ouk; Jun, Sunhong; Lee, Sang Jin; Park, Sungjun; Kosheleva, Irina; Henning, Robert; van Thor, Jasper J; Ihee, Hyotcherl

    2016-03-23

    Real-time probing of structural transitions of a photoactive protein is challenging owing to the lack of a universal time-resolved technique that can probe the changes in both global conformation and light-absorbing chromophores of the protein. In this work, we combine time-resolved X-ray solution scattering (TRXSS) and transient absorption (TA) spectroscopy to investigate how the global conformational changes involved in the photoinduced signal transduction of photoactive yellow protein (PYP) is temporally and spatially related to the local structural change around the light-absorbing chromophore. In particular, we examine the role of internal proton transfer in developing a signaling state of PYP by employing its E46Q mutant (E46Q-PYP), where the internal proton transfer is inhibited by the replacement of a proton donor. The comparison of TRXSS and TA spectroscopy data directly reveals that the global conformational change of the protein, which is probed by TRXSS, is temporally delayed by tens of microseconds from the local structural change of the chromophore, which is probed by TA spectroscopy. The molecular shape of the signaling state reconstructed from the TRXSS curves directly visualizes the three-dimensional conformations of protein intermediates and reveals that the smaller structural change in E46Q-PYP than in wild-type PYP suggested by previous studies is manifested in terms of much smaller protrusion, confirming that the signaling state of E46Q-PYP is only partially developed compared with that of wild-type PYP. This finding provides direct evidence of how the environmental change in the vicinity of the chromophore alters the conformational change of the entire protein matrix. PMID:26960811

  15. Comparative Proteomic Analysis of saccharopolyspora spinosa SP06081 and PR2 strains reveals the differentially expressed proteins correlated with the increase of spinosad yield

    Science.gov (United States)

    2011-01-01

    Background Saccharopolyspora spinosa produces the environment-friendly biopesticide spinosad, a mixture of two polyketide-derived macrolide active ingredients called spinosyns A and D. Therefore considerable interest is in the improvement of spinosad production because of its low yield in wild-type S. spinosa. Recently, a spinosad-hyperproducing PR2 strain with stable heredity was obtained from protoplast regeneration of the wild-type S. spinosa SP06081 strain. A comparative proteomic analysis was performed on the two strains during the first rapid growth phase (RG1) in seed medium (SM) by using label-free quantitative proteomics to investigate the underlying mechanism leading to the enhancement of spinosad yield. Results In total, 224 proteins from the SP06081 strain and 204 proteins from the PR2 strain were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, sharing 140 proteins. A total of 12 proteins directly related to spinosad biosynthesis were identified from the two strains in RG1. Comparative analysis of the shared proteins revealed that approximately 31% of them changed their abundance significantly and fell in all of the functional groups, such as tricarboxylic acid cycles, glycolysis, biosynthetic processes, catabolic processes, transcription, translation, oxidation and reduction. Several key enzymes involved in the synthesis of primary metabolic intermediates used as precursors for spinosad production, energy supply, polyketide chain assembly, deoxysugar methylation, and antioxidative stress were differentially expressed in the same pattern of facilitating spinosad production by the PR2 strain. Real-time reverse transcriptase polymerase chain reaction analysis revealed that four of five selected genes showed a positive correlation between changes at the translational and transcriptional expression level, which further confirmed the proteomic analysis. Conclusions The present study is the first comprehensive and

  16. Conformational changes and ligand recognition of Escherichia coli D-xylose binding protein revealed

    DEFF Research Database (Denmark)

    Sooriyaarachchi, Sanjeewani; Ubhayasekera, Wimal; Park, Chankyu;

    2010-01-01

    ATP binding cassette transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing cognate ligand and directing it to the appropriate membrane permease. Here we report the X-r...

  17. Phosphoproteomics study based on in vivo inhibition reveals sites of calmodulin-dependent protein kinase II regulation in the heart

    NARCIS (Netherlands)

    Scholten, A.; Preisinger, C.; Corradini, E.; Bourgonje, V.J.A.; Hennrich, M.L.; van Veen, T.A.B.; Swaminathan, P.D.; Joiner, M.L.; Vos, M.A.; Anderson, M.E.; Heck, A.J.R.

    2013-01-01

    Background The multifunctional Ca2+‐ and calmodulin‐dependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful

  18. All-atom normal-mode analysis reveals an RNA-induced allostery in a bacteriophage coat protein

    Science.gov (United States)

    Dykeman, Eric C.; Twarock, Reidun

    2010-03-01

    Assembly of the T=3 bacteriophage MS2 is initiated by the binding of a 19 nucleotide RNA stem loop from within the phage genome to a symmetric coat protein dimer. This binding event effects a folding of the FG loop in one of the protein subunits of the dimer and results in the formation of an asymmetric dimer. Since both the symmetric and asymmetric forms of the dimer are needed for the assembly of the protein container, this allosteric switch plays an important role in the life cycle of the phage. We provide here details of an all-atom normal-mode analysis of this allosteric effect. The results suggest that asymmetric contacts between the A -duplex RNA phosphodiester backbone of the stem loop with the EF loop in one coat protein subunit results in an increased dynamic behavior of its FG loop. The four lowest-frequency modes, which encompass motions predominantly on the FG loops, account for over 90% of the increased dynamic behavior due to a localization of the vibrational pattern on a single FG loop. Finally, we show that an analysis of the allosteric effect using an elastic network model fails to predict this localization effect, highlighting the importance of using an all-atom full force field method for this problem.

  19. Molecular Profiling of Synaptic Vesicle Docking Sites Reveals Novel Proteins but Few Differences between Glutamatergic and GABAergic Synapses

    NARCIS (Netherlands)

    Boyken, Janina; Gronborg, Mads; Riedel, Dietmar; Urlaub, Henning; Jahn, Reinhard; Chua, John Jia En

    2013-01-01

    Neurotransmission involves calcium-triggered fusion of docked synaptic vesicles at specialized presynaptic release sites. While many of the participating proteins have been identified, the molecular composition of these sites has not been characterized comprehensively. Here, we report a procedure to

  20. Mass Spectrometry Analysis Coupled with de novo Sequencing Reveals Amino Acid Substitutions in Nucleocapsid Protein from Influenza A Virus

    Directory of Open Access Journals (Sweden)

    Zijian Li

    2014-02-01

    Full Text Available Amino acid substitutions in influenza A virus are the main reasons for both antigenic shift and virulence change, which result from non-synonymous mutations in the viral genome. Nucleocapsid protein (NP, one of the major structural proteins of influenza virus, is responsible for regulation of viral RNA synthesis and replication. In this report we used LC-MS/MS to analyze tryptic digestion of nucleocapsid protein of influenza virus (A/Puerto Rico/8/1934 H1N1, which was isolated and purified by SDS poly-acrylamide gel electrophoresis. Thus, LC-MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three substituted amino acid residues R452K, T423A and N430T in two tryptic peptides. The obtained results provided experimental evidence that amino acid substitutions resulted from non-synonymous gene mutations could be directly characterized by mass spectrometry in proteins of RNA viruses such as influenza A virus.

  1. Global quantitative proteomics reveal up-regulation of endoplasmic reticulum stress response proteins upon depletion of eIF5A in HeLa cells.

    Science.gov (United States)

    Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

    2016-01-01

    The eukaryotic translation factor, eIF5A, is a translation factor essential for protein synthesis, cell growth and animal development. By use of a adenoviral eIF5A shRNA, we have achieved an effective depletion of eIF5A in HeLa cells and undertook in vivo comprehensive proteomic analyses to examine the effects of eIF5A depletion on the total proteome and to identify cellular pathways influenced by eIF5A. The proteome of HeLa cells transduced with eIF5A shRNA was compared with that of scramble shRNA-transduced counterpart by the iTRAQ method. We identified 972 proteins consistently detected in three iTRAQ experiments and 104 proteins with significantly altered levels (protein ratio ≥1.5 or ≤0.66, p-value ≤0.05) at 72 h and/or 96 h of Ad-eIF5A-shRNA transduction. The altered expression levels of key pathway proteins were validated by western blotting. Integration of functional ontology with expression data of the 104 proteins revealed specific biological processes that are prominently up- or down-regulated. Heatmap analysis and Cytoscape visualization of biological networks identified protein folding as the major cellular process affected by depletion of eIF5A. Our unbiased, quantitative, proteomic data demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells. PMID:27180817

  2. "Fluctuograms" reveal the intermittent intra-protein communication in subtilisin Carlsberg and correlate mechanical coupling with co-evolution.

    Directory of Open Access Journals (Sweden)

    Jordi Silvestre-Ryan

    2011-03-01

    Full Text Available The mechanism of intra-protein communication and allosteric coupling is key to understanding the structure-property relationship of protein function. For subtilisin Carlsberg, the Ca²+-binding loop is distal to substrate-binding and active sites, yet the serine protease function depends on Ca²+ binding. The atomic molecular dynamics (MD simulations of apo and Ca²+-bound subtilisin show similar structures and there is no direct evidence that subtilisin has alternative conformations. To model the intra-protein communication due to Ca²+ binding, we transform the sequential segments of an atomic MD trajectory into separate elastic network models to represent anharmonicity and nonlinearity effectively as the temporal and spatial variation of the mechanical coupling network. In analogy to the spectrogram of sound waves, this transformation is termed the "fluctuogram" of protein dynamics. We illustrate that the Ca²+-bound and apo states of subtilisin have different fluctuograms and that intra-protein communication proceeds intermittently both in space and in time. We found that residues with large mechanical coupling variation due to Ca²+ binding correlate with the reported mutation sites selected by directed evolution for improving the stability of subtilisin and its activity in a non-aqueous environment. Furthermore, we utilize the fluctuograms calculated from MD to capture the highly correlated residues in a multiple sequence alignment. We show that in addition to the magnitude, the variance of coupling strength is also an indicative property for the sequence correlation observed in a statistical coupling analysis. The results of this work illustrate that the mechanical coupling networks calculated from atomic details can be used to correlate with functionally important mutation sites and co-evolution.

  3. Proteomic Analysis Reveals Branch-specific Regulation of the Unfolded Protein Response by Nonsense-mediated mRNA Decay.

    Science.gov (United States)

    Sieber, Jana; Hauer, Christian; Bhuvanagiri, Madhuri; Leicht, Stefan; Krijgsveld, Jeroen; Neu-Yilik, Gabriele; Hentze, Matthias W; Kulozik, Andreas E

    2016-05-01

    Nonsense-mediated mRNA decay (NMD) has originally been described as a surveillance mechanism to inhibit the expression of mRNAs with truncated open reading frames (ORFs) and to contribute to the fidelity of gene expression. It is now recognized that NMD also controls the expression of physiological genes with "intact" mRNA. Stress can decrease NMD efficiency and thus increase the mRNA levels of physiological NMD targets. As stress can also inhibit translation, the net outcome for shaping the proteome is difficult to predict. We have thus analyzed de novo protein synthesis in response to NMD inhibition or the induction of mild endoplasmic reticulum (ER) stress by treatment of cells with the reducing agent dithiotreitol (DTT). For this purpose, we combined pulsed azidohomoalanine (AHA) and stable isotope labeling by amino acids in cell culture (SILAC). Labeled proteins were purified by click chemistry-based covalent coupling to agarose beads, trypsinized, fractionated, and analyzed by mass spectrometry (MS). We find that mild ER stress up-regulates the de novo synthesis of components of all three branches of the unfolded protein response (PERK, IRE1 and ATF6) without increasing eIF2α phosphorylation or impairing of protein translation. In contrast, inhibition of NMD induces de novo protein synthesis of downstream targets of the PERK and IRE1 pathways, whereas we could not detect regulation of ATF6-responsive genes. These data thus support a model that implicates a positive feedback loop of ER stress inhibiting NMD efficiency which further promotes the ER stress response in a branch-specific manner. PMID:26896796

  4. Tissue microarray analysis reveals a tight correlation between protein expression pattern and progression of esophageal squamous cell carcinoma

    International Nuclear Information System (INIS)

    The development of esophageal squamous cell carcinoma (ESCC) progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS) and carcinoma in situ (CIS), subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression. Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5γ2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay. In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5γ2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were 'early' corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, 'intermediate' to severe DYS and CIS with overexpression of FADD and CK14, and 'late' to invasive lesions (ESCC) and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5γ2 and SPARC. Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC

  5. Promoter Analysis Reveals Globally Differential Regulation of Human Long Non-Coding RNA and Protein-Coding Genes

    KAUST Repository

    Alam, Tanvir

    2014-10-02

    Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as protein-coding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptional regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future.

  6. Tissue microarray analysis reveals a tight correlation between protein expression pattern and progression of esophageal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    He Zu-gen

    2006-12-01

    Full Text Available Abstract Background The development of esophageal squamous cell carcinoma (ESCC progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS and carcinoma in situ (CIS, subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression. Methods Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5γ2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay. Results In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5γ2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5γ2 and SPARC. Conclusion Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC.

  7. Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli.

    Science.gov (United States)

    Srivastava, Aashish; Asahara, Haruichi; Zhang, Meng; Zhang, Weijia; Liu, Haiying; Cui, Sheng; Jin, Qi; Chong, Shaorong

    2016-01-01

    Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis), purified tRNAs and ribosomes from Mycobacterium smegmatis (M. smegmatis), and an aminoacyl-tRNA synthetase (AARS) mixture from the cell-extract of M. smegmatis. We demonstrate that such mycobacterial translation system was efficient in in vitro protein synthesis, and enabled functional comparisons of translational components between the gram-positive Mycobacterium and the gram-negative E. coli. Although mycobacterial translation factors and ribosomes were highly compatible with their E. coli counterparts, M. smegmatis tRNAs were not properly charged by the E. coli AARSs to allow efficient translation of a reporter. In contrast, both E. coli and M. smegmatis tRNAs exhibited similar activity with the semi-purified M. smegmatis AARSs mixture for in vitro translation. We further demonstrated the use of both mycobacterial and E. coli translation systems as comparative in vitro assays for small-molecule antibiotics that target protein translation. While mycobacterial and E. coli translation were both inhibited at the same IC50 by the antibiotic spectinomycin, mycobacterial translation was preferentially inhibited by the antibiotic tetracycline, suggesting that there may be structural differences at the antibiotic binding sites between the ribosomes of Mycobacterium and E. coli. Our results illustrate an alternative approach for antibiotic discovery and functional studies of protein translation in mycobacteria and possibly other bacterial pathogens. PMID:27564552

  8. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

    Energy Technology Data Exchange (ETDEWEB)

    Saikatendu, Kumar Singh; Zhang, Xuejun; Kinch, Lisa; Leybourne, Matthew; Grishin, Nick V.; Zhang, Hong (Texas-D); (U. of Texas-SMED)

    2009-01-26

    The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. The structure of SA1388 has been solved to 2.0{angstrom} resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric 'lids' formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric

  9. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

    International Nuclear Information System (INIS)

    The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. The structure of SA1388 has been solved to 2.0(angstrom) resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric 'lids' formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric 'lids' that

  10. Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer

    Directory of Open Access Journals (Sweden)

    Nelson Laura D

    2012-06-01

    Full Text Available Abstract Background Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics. Methods Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman’s rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts. Results Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p = 0.024. EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated

  11. Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

    Energy Technology Data Exchange (ETDEWEB)

    Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.; (Duke)

    2010-05-25

    Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T. maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of {beta}(1 {yields} 4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.

  12. Uncovering SUMOylation Dynamics during Cell-Cycle Progression Reveals FoxM1 as a Key Mitotic SUMO Target Protein

    DEFF Research Database (Denmark)

    Schimmel, Joost; Eifler, Karolin; Sigurdsson, Jón Otti; Cuijpers, Sabine A G; Hendriks, Ivo A; Verlaan-de Vries, Matty; Kelstrup, Christian D; Francavilla, Chiara; Medema, René H; Olsen, Jesper Velgaard; Vertegaal, Alfred C O

    2014-01-01

    Loss of small ubiquitin-like modification (SUMOylation) in mice causes genomic instability due to the missegregation of chromosomes. Currently, little is known about the identity of relevant SUMO target proteins that are involved in this process and about global SUMOylation dynamics during cell-cycle...... progression. We performed a large-scale quantitative proteomics screen to address this and identified 593 proteins to be SUMO-2 modified, including the Forkhead box transcription factor M1 (FoxM1), a key regulator of cell-cycle progression and chromosome segregation. SUMOylation of FoxM1 peaks during G2 and M...... relieving FoxM1 autorepression. Cells deficient for FoxM1 SUMOylation showed increased levels of polyploidy. Our findings contribute to understanding the role of SUMOylation during cell-cycle progression....

  13. Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong (Cornell); (NWU)

    2012-05-22

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.

  14. Identification of a Novel Nonstructural Protein, VP9, from White Spot Syndrome Virus: Its Structure Reveals a Ferredoxin Fold with Specific Metal Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Liu,Y.; Wu, J.; Song, J.; Sivaraman, J.; Hew, C.

    2006-01-01

    White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP9, a full-length protein of WSSV, encoded by open reading frame wsv230, was identified for the first time in the infected Penaeus monodon shrimp tissues, gill, and stomach as a novel, nonstructural protein by Western blotting, mass spectrometry, and immunoelectron microscopy. Real-time reverse transcription-PCR demonstrated that the transcription of VP9 started from the early to the late stage of WSSV infection as a major mRNA species. The structure of full-length VP9 was determined by both X-ray and nuclear magnetic resonance (NMR) techniques. It is the first structure to be reported for WSSV proteins. The crystal structure of VP9 revealed a ferredoxin fold with divalent metal ion binding sites. Cadmium sulfate was found to be essential for crystallization. The Cd2+ ions were bound between the monomer interfaces of the homodimer. Various divalent metal ions have been titrated against VP9, and their interactions were analyzed using NMR spectroscopy. The titration data indicated that VP9 binds with both Zn2+ and Cd2+. VP9 adopts a similar fold as the DNA binding domain of the papillomavirus E2 protein. Based on our present investigations, we hypothesize that VP9 might be involved in the transcriptional regulation of WSSV, a function similar to that of the E2 protein during papillomavirus infection of the host cells.

  15. Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs

    International Nuclear Information System (INIS)

    The structure of VapB, a member of the Vap protein family that is involved in virulence of the bacterial pathogen R. equi, was determined by SAD phasing and reveals an eight-stranded antiparallel β-barrel similar to avidin, suggestive of a binding function. Made up of two Greek-key motifs, the topology of VapB is unusual or even unique. Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology

  16. Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs

    Energy Technology Data Exchange (ETDEWEB)

    Geerds, Christina [Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany); Wohlmann, Jens; Haas, Albert [University of Bonn, Ulrich-Haberland Strasse 61a, 53121 Bonn (Germany); Niemann, Hartmut H., E-mail: hartmut.niemann@uni-bielefeld.de [Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany)

    2014-06-18

    The structure of VapB, a member of the Vap protein family that is involved in virulence of the bacterial pathogen R. equi, was determined by SAD phasing and reveals an eight-stranded antiparallel β-barrel similar to avidin, suggestive of a binding function. Made up of two Greek-key motifs, the topology of VapB is unusual or even unique. Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology.

  17. Improved tag-switch method reveals that thioredoxin acts as depersulfidase and controls the intracellular levels of protein persulfidation

    OpenAIRE

    Wedmann, R.; Onderka, C.; Wei, S; Szijarto, I.A.; Miljkovic, J.L.; Mitrovic, A; Lange, M.; Savitsky, S.; Yadav, P.K.; Torregrossa, R.; Harrer, E.G.; Harrer, T; Ishii, I.; Gollasch, M.; Wood, M.E.

    2016-01-01

    Hydrogen sulfide (H2S) has emerged as a signalling molecule capable of regulating several important physiological functions such as blood pressure, neurotransmission and inflammation. The mechanisms behind these effects are still largely elusive and oxidative posttranslational modification of cysteine residues (protein persulfidation or S-sulfhydration) has been proposed as the main pathway for H2S-induced biological and pharmacological effects. As a signalling mechanism, persulfidation has t...

  18. Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

    OpenAIRE

    Allison Marie Barbaglia; Banita eTamot; Veronica eGreve; Susanne eHoffmann-Benning

    2016-01-01

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identifi...

  19. Phloem Proteomics Reveals New Lipid-Binding Proteins with a Putative Role in Lipid-Mediated Signaling

    OpenAIRE

    Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-01-01

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identifi...

  20. Combined proteomics and pathways analysis of collecting duct reveals a protein regulatory network activated in vasopressin escape

    OpenAIRE

    Hoorn, Ewout J; Hoffert, Jason D.; Knepper, Mark A.

    2005-01-01

    Low sensitivity is characteristic of many proteomics methods. Here we present an approach that combines proteomics based on “Difference Gel Electrophoresis” (DIGE) with bioinformatic pathways analysis to identify both abundant and relatively non-abundant proteins in inner medullary collecting duct (IMCD) altered in abundance during escape from vasopressin-induced antidiuresis. Rats received the vasopressin analog dDAVP by osmotic minipump plus either a daily water load (vasopressin escape) or...

  1. Phosphoproteome Analysis of Functional Mitochondria Isolated from Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes*

    OpenAIRE

    Zhao, Xiaolu; León, Ileana R.; Bak, Steffen; Mogensen, Martin; Wrzesinski, Krzysztof; Højlund, Kurt; Jensen, Ole Nørregaard

    2010-01-01

    Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human...

  2. Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia

    OpenAIRE

    Julie B. McLean; Moylan, Jennifer S.; Horrell, Erin M. W.; Andrade, Francisco H.

    2015-01-01

    Aims: At the time of diagnosis, 60% of lung cancer patients present with cachexia, a severe wasting syndrome that increases morbidity and mortality. Tumors secrete multiple factors that contribute to cachectic muscle wasting, and not all of these factors have been identified. We used Orbitrap electrospray ionization mass spectrometry to identify novel cachexia-inducing candidates in media conditioned with Lewis lung carcinoma cells (LCM). Results: One-hundred and 58 proteins were confirmed in...

  3. The Short-Lived Signaling State of the Photoactive Yellow Protein Photoreceptor Revealed by Combined Structural Probes

    OpenAIRE

    Ramachandran, Pradeep L.; Lovett, Janet E.; Carl, Patrick J.; Cammarata, Marco; Lee, Jae Hyuk; Jung, Yang Ouk; Ihee, Hyotcherl; Timmel, Christiane R.; Jasper J. van Thor

    2011-01-01

    The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron ...

  4. A functional study of the conserved LSM proteins in C. elegans reveals their involvement in the stress response of metazoans

    OpenAIRE

    Cornes Maragliano, Eric, 1987-

    2015-01-01

    Material addicional: http://hdl.handle.net/10230/25608 Lsm proteins regulate RNA metabolism and are conserved in the three domains of life, typically functioning as RNA-binding complexes involved in a wide range of post-transcriptional mechanisms. Generally, their functions have been explored in unicellular models using biochemical approaches; however their physiological roles in multicellular organisms remain unknown. This gap in knowledge is biomedically relevant since alterations of ind...

  5. Differences in abundances of cell-signalling proteins in blood reveal novel biomarkers for early detection of clinical Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Mateus Rocha de Paula

    Full Text Available BACKGROUND: In November 2007 a study published in Nature Medicine proposed a simple test based on the abundance of 18 proteins in blood to predict the onset of clinical symptoms of Alzheimer's Disease (AD two to six years before these symptoms manifest. Later, another study, published in PLoS ONE, showed that only five proteins (IL-1, IL-3, EGF, TNF- and G-CSF have overall better prediction accuracy. These classifiers are based on the abundance of 120 proteins. Such values were standardised by a Z-score transformation, which means that their values are relative to the average of all others. METHODOLOGY: The original datasets from the Nature Medicine paper are further studied using methods from combinatorial optimisation and Information Theory. We expand the original dataset by also including all pair-wise differences of z-score values of the original dataset ("metafeatures". Using an exact algorithm to solve the resulting Feature Set problem, used to tackle the feature selection problem, we found signatures that contain either only features, metafeatures or both, and evaluated their predictive performance on the independent test set. CONCLUSIONS: It was possible to show that a specific pattern of cell signalling imbalance in blood plasma has valuable information to distinguish between NDC and AD samples. The obtained signatures were able to predict AD in patients that already had a Mild Cognitive Impairment (MCI with up to 84% of sensitivity, while maintaining also a strong prediction accuracy of 90% on a independent dataset with Non Demented Controls (NDC and AD samples. The novel biomarkers uncovered with this method now confirms ANG-2, IL-11, PDGF-BB, CCL15/MIP-1; and supports the joint measurement of other signalling proteins not previously discussed: GM-CSF, NT-3, IGFBP-2 and VEGF-B.

  6. Impaired Macromolecular Protein Pools in Fronto-Striato-Thalamic Circuits in Type 2 Diabetes Revealed by Magnetization Transfer Imaging

    OpenAIRE

    Yang, Shaolin; Ajilore, Olusola; Wu, Minjie; Lamar, Melissa; Kumar, Anand

    2014-01-01

    Previous research has shown that type 2 diabetes mellitus (T2DM) is associated with white matter microstructural changes, cognitive impairment, and decreased resting-state functional connectivity and spontaneous brain activity. This study used magnetization transfer imaging to examine, for the first time, the integrity of macromolecular protein pools in fronto-striato-thalamic circuits and its clinical and cognitive correlates in patients with T2DM. T2DM patients without mood disorders (n = 2...

  7. Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel.

    Science.gov (United States)

    Zhao, Haiyan; Speir, Jeffrey A; Matsui, Tsutomu; Lin, Zihan; Liang, Lingfei; Lynn, Anna Y; Varnado, Brittany; Weiss, Thomas M; Tang, Liang

    2016-01-01

    The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. The gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device. PMID:26882199

  8. Confined Diffusion Without Fences of a G-Protein-Coupled Receptor as Revealed by Single Particle Tracking

    Science.gov (United States)

    Daumas, Frédéric; Destainville, Nicolas; Millot, Claire; Lopez, André; Dean, David; Salomé, Laurence

    2003-01-01

    Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the μ-opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients. PMID:12524289

  9. Transcriptomic analysis reveals numerous diverse protein kinases and transcription factors involved in desiccation tolerance in the resurrection plant Myrothamnus flabellifolia.

    Science.gov (United States)

    Ma, Chao; Wang, Hong; Macnish, Andrew J; Estrada-Melo, Alejandro C; Lin, Jing; Chang, Youhong; Reid, Michael S; Jiang, Cai-Zhong

    2015-01-01

    The woody resurrection plant Myrothamnus flabellifolia has remarkable tolerance to desiccation. Pyro-sequencing technology permitted us to analyze the transcriptome of M. flabellifolia during both dehydration and rehydration. We identified a total of 8287 and 8542 differentially transcribed genes during dehydration and rehydration treatments respectively. Approximately 295 transcription factors (TFs) and 484 protein kinases (PKs) were up- or down-regulated in response to desiccation stress. Among these, the transcript levels of 53 TFs and 91 PKs increased rapidly and peaked early during dehydration. These regulators transduce signal cascades of molecular pathways, including the up-regulation of ABA-dependent and independent drought stress pathways and the activation of protective mechanisms for coping with oxidative damage. Antioxidant systems are up-regulated, and the photosynthetic system is modified to reduce ROS generation. Secondary metabolism may participate in the desiccation tolerance of M. flabellifolia as indicated by increases in transcript abundance of genes involved in isopentenyl diphosphate biosynthesis. Up-regulation of genes encoding late embryogenesis abundant proteins and sucrose phosphate synthase is also associated with increased tolerance to desiccation. During rehydration, the transcriptome is also enriched in transcripts of genes encoding TFs and PKs, as well as genes involved in photosynthesis, and protein synthesis. The data reported here contribute comprehensive insights into the molecular mechanisms of desiccation tolerance in M. flabellifolia. PMID:26504577

  10. Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria.

    Science.gov (United States)

    Schorey, J S; Holsti, M A; Ratliff, T L; Allen, P M; Brown, E J

    1996-07-01

    Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium, we have cloned the M. avium fibronectin-attachment protein (FAP). The M. avium FAP (FAP-A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Mycobacterium leprae and Mycobacterium tuberculosis. Using recombinant FAP-A and FAP-A peptides, we show that two non-continuous regions in FAP-A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette-Guerin (BCG). These regions have no homology to eukaryotic fibronectin-binding proteins and are only distantly related to fibronectin-binding peptides of Gram-positive bacteria. Nevertheless, these fibronectin-binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts. PMID:8858587

  11. Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel.

    Directory of Open Access Journals (Sweden)

    Haiyan Zhao

    Full Text Available The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. The gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.

  12. Multi-species protein similarity clustering reveals novel expanded immune gene families in the eastern oyster Crassostrea virginica.

    Science.gov (United States)

    McDowell, Ian C; Modak, Tejashree H; Lane, Chris E; Gomez-Chiarri, Marta

    2016-06-01

    Comparative genomics research in non-model species has highlighted how invertebrate hosts possess complex diversified repertoires of immune molecules. The levels of diversification in particular immune gene families appear to differ between invertebrate lineages and even between species within lineages, reflecting differences not only in evolutionary histories, but also in life histories, environmental niches, and pathogen exposures. The goal of this research was to identify immune-related gene families experiencing high levels of diversification in eastern oysters, Crassostrea virginica. Families containing 1) transcripts differentially expressed in eastern oysters in response to bacterial challenge and 2) a larger number of transcripts compared to other species included those coding for the C1q and C-type lectin domain containing proteins (C1qDC and CTLDC), GTPase of the immune-associated proteins (GIMAP), scavenger receptors (SR), fibrinogen-C domain containing proteins (also known as FREPs), dopamine beta-hydrolase (DBH), interferon-inducible 44 (IFI44), serine protease inhibitors, apextrin, and dermatopontin. Phylogenetic analysis of two of the families significantly expanded in bivalves, IFI44 and GIMAP, showed a patchy distribution within both protostomes and deuterostomes, suggesting multiple independent losses and lineage-specific expansions. Increased availability of genomic information for a broader range of non-model species broadly distributed through vertebrate and invertebrate phyla will likely lead to improved knowledge on mechanisms of immune-gene diversification. PMID:27033806

  13. Activity-Based Protein Profiling Reveals Mitochondrial Oxidative Enzyme Impairment and Restoration in Diet-Induced Obese Mice

    Energy Technology Data Exchange (ETDEWEB)

    Sadler, Natalie C.; Angel, Thomas E.; Lewis, Michael P.; Pederson, Leeanna M.; Chauvigne-Hines, Lacie M.; Wiedner, Susan D.; Zink, Erika M.; Smith, Richard D.; Wright, Aaron T.

    2012-10-24

    High-fat diet (HFD) induced obesity and concomitant development of insulin resistance (IR) and type 2 diabetes mellitus have been linked to mitochondrial dysfunction. However, it is not clear whether mitochondrial dysfunction is a direct effect of a HFD or if the mitochondrial function is reduced with increased HFD duration. We hypothesized that the function of mitochondrial oxidative and lipid metabolism functions in skeletal muscle mitochondria for HFD mice are similar or elevated relative to standard diet (SD) mice, thereby IR is neither cause nor consequence of mitochondrial dysfunction. We applied a chemical probe approach to identify functionally reactive ATPases and nucleotide-binding proteins in mitochondria isolated from skeletal muscle of C57Bl/6J mice fed HFD or SD chow for 2-, 8-, or 16-weeks; feeding time points known to induce IR. A total of 293 probe-labeled proteins were identified by mass spectrometry-based proteomics, of which 54 differed in abundance between HFD and SD mice. We found proteins associated with the TCA cycle, oxidative phosphorylation (OXPHOS), and lipid metabolism were altered in function when comparing SD to HFD fed mice at 2-weeks, however by 16-weeks HFD mice had TCA cycle, β-oxidation, and respiratory chain function at levels similar to or higher than SD mice.

  14. Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall

    Directory of Open Access Journals (Sweden)

    Monica eDe Caroli

    2015-08-01

    Full Text Available The plant endomembrane system is massively involved in the synthesis, transport and secretion of cell wall polysaccharides and proteins; however, the molecular mechanisms underlying trafficking toward the apoplast are largely unknown. Besides constitutive, the existence of a regulated secretory pathway has been proposed. A polygalacturonase inhibitor protein (PGIP2, known to move as soluble cargo and reach the cell wall through a mechanism distinguishable from default, was dissected in its main functional domains (A, B, C, D, and C sub-fragments (C1-10, to identify signals essential for its regulated targeting. The secretion patterns of the fluorescent chimeras obtained by fusing different PGIP2 domains to the green fluorescent protein (GFP were analyzed. PGIP2 N-terminal and leucine-rich repeat domains (B and C, respectively seem to operate as holding/releasing signals, respectively, during PGIP2 transit through the Golgi. The B domain slows down PGIP2 secretion by transiently interacting with Golgi membranes. Its depletion leads, in fact, to the secretion via default (Sp2-susceptible of the ACD-GFP chimera faster than PGIP2. Depending on its length (at least the first 5 leucine-rich repeats are required, the C domain modulates B interaction with Golgi membranes allowing the release of chimeras and their extracellular secretion through a Sp2 independent pathway. The addition of the vacuolar sorting determinant Chi to PGIP2 diverts the path of the protein from cell wall to vacuole, suggesting that C domain is a releasing rather than a cell wall sorting signal.

  15. Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

    Science.gov (United States)

    Shah, Punit; Wang, Xiangchun; Yang, Weiming; Toghi Eshghi, Shadi; Sun, Shisheng; Hoti, Naseruddin; Chen, Lijun; Yang, Shuang; Pasay, Jered; Rubin, Abby; Zhang, Hui

    2015-10-01

    Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in

  16. MALDI-mass spectrometric imaging revealing hypoxia-driven lipids and proteins in a breast tumor model

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jiang; Chughtai, Kamila; Purvine, Samuel O.; Bhujwalla, Zaver M.; Raman, Venu; Pasa-Tolic, Ljiljana; Heeren, Ronald M.; Glunde, Kristine

    2015-06-16

    Hypoxic areas are a common feature of rapidly growing malignant tumors and their metastases, and are typically spatially heterogeneous. Hypoxia has a strong impact on tumor cell biology and contributes to tumor progression in multiple ways. To date, only a few molecular key players in tumor hypoxia, such as for example hypoxia-inducible factor-1 (HIF-1), have been discovered. The distribution of biomolecules is frequently heterogeneous in the tumor volume, and may be driven by hypoxia and HIF-1α. Understanding the spatially heterogeneous hypoxic response of tumors is critical. Mass spectrometric imaging (MSI) provides a unique way of imaging biomolecular distributions in tissue sections with high spectral and spatial resolution. In this paper, breast tumor xenografts grown from MDA-MB-231-HRE-tdTomato cells, with a red fluorescent tdTomato protein construct under the control of a hypoxia response element (HRE)-containing promoter driven by HIF-1α, were used to detect the spatial distribution of hypoxic regions. We elucidated the 3D spatial relationship between hypoxic regions and the localization of small molecules, metabolites, lipids, and proteins by using principal component analysis – linear discriminant analysis (PCA-LDA) on 3D rendered MSI volume data from MDA-MB-231-HRE-tdTomato breast tumor xenografts. In this study we identified hypoxia-regulated proteins active in several distinct pathways such as glucose metabolism, regulation of actin cytoskeleton, protein folding, translation/ribosome, splicesome, the PI3K-Akt signaling pathway, hemoglobin chaperone, protein processing in endoplasmic reticulum, detoxification of reactive oxygen species, aurora B signaling/apoptotic execution phase, the RAS signaling pathway, the FAS signaling pathway/caspase cascade in apoptosis and telomere stress induced senescence. In parallel we also identified co-localization of hypoxic regions and various lipid species such as PC(16:0/18:1), PC(16:0/18:2), PC(18:0/18:1), PC

  17. Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella Typimurium in response to infection-like conditions

    Energy Technology Data Exchange (ETDEWEB)

    Ansong, Charles; Wu, Si; Meng, Da; Liu, Xiaowen; Brewer, Heather M.; Kaiser, Brooke LD; Nakayasu, Ernesto S.; Cort, John R.; Pevzner, Pavel A.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.; Pasa-Tolic, Ljiljana

    2013-06-18

    Characterization of the mature protein complement in cells is crucial for a better understanding of cellular processes on a systems-wide scale. Bottom-up proteomic approaches often lead to loss of critical information about an endogenous protein’s actual state due to post translational modifications (PTMs) and other processes. Top-down approaches that involve analysis of the intact protein can address this concern but present significant analytical challenges related to the separation quality needed, measurement sensitivity, and speed that result in low throughput and limited coverage. Here we used single-dimension ultra high pressure liquid chromatography mass spectrometry to investigate the comprehensive ‘intact’ proteome of the Gram negative bacterial pathogen Salmonella Typhimurium. Top-down proteomics analysis revealed 563 unique proteins including 1665 proteoforms generated by PTMs, representing the largest microbial top-down dataset reported to date. Our analysis not only confirmed several previously recognized aspects of Salmonella biology and bacterial PTMs in general, but also revealed several novel biological insights. Of particular interest was differential utilization of the protein S-thiolation forms S-glutathionylation and S-cysteinylation in response to infection-like conditions versus basal conditions, which was corroborated by changes in corresponding biosynthetic pathways. This differential utilization highlights underlying metabolic mechanisms that modulate changes in cellular signaling, and represents to our knowledge the first report of S-cysteinylation in Gram negative bacteria. The demonstrated utility of our simple proteome-wide intact protein level measurement strategy for gaining biological insight should promote broader adoption and applications of top-down proteomics approaches.

  18. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  19. The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

    Science.gov (United States)

    Vivancos, Julien; Spinner, Lara; Mazubert, Christelle; Charlot, Florence; Paquet, Nicolas; Thareau, Vincent; Dron, Michel; Nogué, Fabien; Charon, Céline

    2012-03-01

    The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM. PMID:22170036

  20. In vivo NMR field-cycling relaxation spectroscopy reveals 14N1H relaxation sinks in the backbones of proteins

    International Nuclear Information System (INIS)

    In this preliminary note, the authors report an in vivo study of Hirudo medicinalis, using field-cycling relaxation spectroscopy, showing clear 14N1H quadrupole dips, proving that the amide 14N1H groups of proteins can act as relaxation sinks in a frequency range relevant for NMR tomography. Also, as a byproduct of this work it is noted that during these experiments, leeches were exposed to field variation rates of about 50 Ts-1 in several thousand field-cycles up and down, without any obvious damage. (U.K.)

  1. Unique features of odorant-binding proteins of the parasitoid wasp Nasonia vitripennis revealed by genome annotation and comparative analyses.

    Directory of Open Access Journals (Sweden)

    Filipe G Vieira

    Full Text Available Insects are the most diverse group of animals on the planet, comprising over 90% of all metazoan life forms, and have adapted to a wide diversity of ecosystems in nearly all environments. They have evolved highly sensitive chemical senses that are central to their interaction with their environment and to communication between individuals. Understanding the molecular bases of insect olfaction is therefore of great importance from both a basic and applied perspective. Odorant binding proteins (OBPs are some of most abundant proteins found in insect olfactory organs, where they are the first component of the olfactory transduction cascade, carrying odorant molecules to the olfactory receptors. We carried out a search for OBPs in the genome of the parasitoid wasp Nasonia vitripennis and identified 90 sequences encoding putative OBPs. This is the largest OBP family so far reported in insects. We report unique features of the N. vitripennis OBPs, including the presence and evolutionary origin of a new subfamily of double-domain OBPs (consisting of two concatenated OBP domains, the loss of conserved cysteine residues and the expression of pseudogenes. This study also demonstrates the extremely dynamic evolution of the insect OBP family: (i the number of different OBPs can vary greatly between species; (ii the sequences are highly diverse, sometimes as a result of positive selection pressure with even the canonical cysteines being lost; (iii new lineage specific domain arrangements can arise, such as the double domain OBP subfamily of wasps and mosquitoes.

  2. Quantitative Proteomic Analysis of Mitochondrial Proteins Reveals Pro-Survival Mechanisms in the Perpetuation of Radiation-Induced Genomic Instability

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, Stefani N.; Waters, Katrina M.; Morgan, William F.; Yang, Austin; Baulch, Janet E.

    2012-07-26

    Radiation induced genomic instability is a well-studied phenomenon that is measured as mitotically heritable genetic alterations observed in the progeny of an irradiated cell. The mechanisms that perpetuate this instability are unclear, however, a role for chronic oxidative stress has consistently been demonstrated. In the chromosomally unstable LS12 cell line, oxidative stress and genomic instability were correlated with mitochondrial dysfunction. To clarify this mitochondrial dysfunction and gain insight into the mechanisms underlying radiation induced genomic instability we have evaluated the mitochondrial sub-proteome and performed quantitative mass spectrometry (MS) analysis of LS12 cells. Of 98 quantified mitochondrial proteins, 17 met criteria for fold changes and reproducibility; and 11 were statistically significant in comparison with the stable parental GM10115 cell line. Previous observations implicated defects in the electron transport chain (ETC) in the LS12 cell mitochondrial dysfunction. Proteomic analysis supports these observations, demonstrating significantly reduced levels of mitochondrial cytochrome c, the intermediary between complexes III and IV of the ETC. Results also suggest that LS12 cells compensate for ETC dysfunction and oxidative stress through increased levels of tricarboxylic acid cycle enzymes and up-regulation of proteins that protect against oxidative stress and apoptosis. More than one cellular defect is likely to contribute to the genomic instability phenotype. These data suggest that LS12 cells have adapted mechanisms that allow survival under sub-optimal conditions of oxidative stress and compromised mitochondrial function to perpetuate genomic instability.

  3. Subcellular fractionation and localization studies reveal a direct interaction of the fragile X mental retardation protein (FMRP with nucleolin.

    Directory of Open Access Journals (Sweden)

    Mohamed S Taha

    Full Text Available Fragile X mental Retardation Protein (FMRP is a well-known regulator of local translation of its mRNA targets in neurons. However, despite its ubiquitous expression, the role of FMRP remains ill-defined in other cell types. In this study we investigated the subcellular distribution of FMRP and its protein complexes in HeLa cells using confocal imaging as well as detergent-free fractionation and size exclusion protocols. We found FMRP localized exclusively to solid compartments, including cytosolic heavy and light membranes, mitochondria, nuclear membrane and nucleoli. Interestingly, FMRP was associated with nucleolin in both a high molecular weight ribosomal and translation-associated complex (≥6 MDa in the cytosol, and a low molecular weight complex (∼200 kDa in the nucleoli. Consistently, we identified two functional nucleolar localization signals (NoLSs in FMRP that are responsible for a strong nucleolar colocalization of the C-terminus of FMRP with nucleolin, and a direct interaction of the N-terminus of FMRP with the arginine-glycine-glycine (RGG domain of nucleolin. Taken together, we propose a novel mechanism by which a transient nucleolar localization of FMRP underlies a strong nucleocytoplasmic translocation, most likely in a complex with nucleolin and possibly ribosomes, in order to regulate translation of its target mRNAs.

  4. Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization in Entamoeba histolytica.

    Science.gov (United States)

    Perdomo, Doranda; Manich, Maria; Syan, Sylvie; Olivo-Marin, Jean-Christophe; Dufour, Alexandre C; Guillén, Nancy

    2016-08-01

    The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well-defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER-Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin-rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica. PMID:26857352

  5. Comparative proteomics of root plasma membrane proteins reveals the involvement of calcium signalling in NaCl-facilitated nitrate uptake in Salicornia europaea.

    Science.gov (United States)

    Nie, Lingling; Feng, Juanjuan; Fan, Pengxiang; Chen, Xianyang; Guo, Jie; Lv, Sulian; Bao, Hexigeduleng; Jia, Weitao; Tai, Fang; Jiang, Ping; Wang, Jinhui; Li, Yinxin

    2015-08-01

    Improving crop nitrogen (N) use efficiency under salinity is essential for the development of sustainable agriculture in marginal lands. Salicornia europaea is a succulent euhalophyte that can survive under high salinity and N-deficient habitat conditions, implying that a special N assimilation mechanism may exist in this plant. In this study, phenotypic and physiological changes of S. europaea were investigated under different nitrate and NaCl levels. The results showed that NaCl had a synergetic effect with nitrate on the growth of S. europaea. In addition, the shoot nitrate concentration and nitrate uptake rate of S. europaea were increased by NaCl treatment under both low N and high N conditions, suggesting that nitrate uptake in S. europaea was NaCl facilitated. Comparative proteomic analysis of root plasma membrane (PM) proteins revealed 81 proteins, whose abundance changed significantly in response to NaCl and nitrate. These proteins are involved in metabolism, cell signalling, transport, protein folding, membrane trafficking, and cell structure. Among them, eight proteins were calcium signalling components, and the accumulation of seven of the above-mentioned proteins was significantly elevated by NaCl treatment. Furthermore, cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was significantly elevated in S. europaea under NaCl treatment. The application of the Ca(2+) channel blocker LaCl3 not only caused a decrease in nitrate uptake rate, but also attenuated the promoting effects of NaCl on nitrate uptake rates. Based on these results, a possible regulatory network of NaCl-facilitated nitrate uptake in S. europaea focusing on the involvement of Ca(2+) signalling was proposed. PMID:25956883

  6. Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function during Nanoparticle-Induced Oxidative Stress.

    Science.gov (United States)

    Duan, Jicheng; Kodali, Vamsi K; Gaffrey, Matthew J; Guo, Jia; Chu, Rosalie K; Camp, David G; Smith, Richard D; Thrall, Brian D; Qian, Wei-Jun

    2016-01-26

    Engineered nanoparticles (ENPs) are increasingly utilized for commercial and medical applications; thus, understanding their potential adverse effects is an important societal issue. Herein, we investigated protein S-glutathionylation (SSG) as an underlying regulatory mechanism by which ENPs may alter macrophage innate immune functions, using a quantitative redox proteomics approach for site-specific measurement of SSG modifications. Three high-volume production ENPs (SiO2, Fe3O4, and CoO) were selected as representatives which induce low, moderate, and high propensity, respectively, to stimulate cellular reactive oxygen species (ROS) and disrupt macrophage function. The SSG modifications identified highlighted a broad set of redox sensitive proteins and specific Cys residues which correlated well with the overall level of cellular redox stress and impairment of macrophage phagocytic function (CoO > Fe3O4 ≫ SiO2). Moreover, our data revealed pathway-specific differences in susceptibility to SSG between ENPs which induce moderate versus high levels of ROS. Pathways regulating protein translation and protein stability indicative of ER stress responses and proteins involved in phagocytosis were among the most sensitive to SSG in response to ENPs that induce subcytoxic levels of redox stress. At higher levels of redox stress, the pattern of SSG modifications displayed reduced specificity and a broader set pathways involving classical stress responses and mitochondrial energetics (e.g., glycolysis) associated with apoptotic mechanisms. An important role for SSG in regulation of macrophage innate immune function was also confirmed by RNA silencing of glutaredoxin, a major enzyme which reverses SSG modifications. Our results provide unique insights into the protein signatures and pathways that serve as ROS sensors and may facilitate cellular adaption to ENPs, versus intracellular targets of ENP-induced oxidative stress that are linked to irreversible cell outcomes. PMID

  7. Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene

    Directory of Open Access Journals (Sweden)

    McGuire John J

    2005-09-01

    Full Text Available Abstract Background The rTS gene (ENOSF1, first identified in Homo sapiens as a gene complementary to the thymidylate synthase (TYMS mRNA, is known to encode two protein isoforms, rTSα and rTSβ. The rTSβ isoform appears to be an enzyme responsible for the synthesis of signaling molecules involved in the down-regulation of thymidylate synthase, but the exact cellular functions of rTS genes are largely unknown. Results Through comparative genomic sequence analysis, we predicted the existence of a novel protein isoform, rTS, which has a 27 residue longer N-terminus by virtue of utilizing an alternative start codon located upstream of the start codon in rTSβ. We observed that a similar extended N-terminus could be predicted in all rTS genes for which genomic sequences are available and the extended regions are conserved from bacteria to human. Therefore, we reasoned that the protein with the extended N-terminus might represent an ancestral form of the rTS protein. Sequence analysis strongly predicts a mitochondrial signal sequence in the extended N-terminal of human rTSγ, which is absent in rTSβ. We confirmed the existence of rTS in human mitochondria experimentally by demonstrating the presence of both rTSγ and rTSβ proteins in mitochondria isolated by subcellular fractionation. In addition, our comprehensive analysis of rTS orthologous sequences reveals an unusual phylogenetic distribution of this gene, which suggests the occurrence of one or more horizontal gene transfer events. Conclusion The presence of two rTS isoforms in mitochondria suggests that the rTS signaling pathway may be active within mitochondria. Our report also presents an example of identifying novel protein isoforms and for improving gene annotation through comparative genomic analysis.

  8. Constitutive Activation of the Fission Yeast Pheromone-Responsive Pathway Induces Ectopic Meiosis and Reveals Ste11 as a Mitogen-Activated Protein Kinase Target

    DEFF Research Database (Denmark)

    Kjærulff, Søren; Lautrup-Larsen, I.; Truelsen, S.;

    2005-01-01

    In the fission yeast Schizosaccharomyces pombe, meiosis normally takes place in diploid zygotes resulting from conjugation of haploid cells. In the present study, we report that the expression of a constitutively activated version of the pheromone-responsive mitogen-activated protein kinase kinase...... kinase (MAP3K) Byr2 can induce ectopic meiosis directly in haploid cells. We find that the Ste11 transcription factor becomes constitutively expressed in these cells and that the expression of pheromone-responsive genes no longer depends on nitrogen starvation. Epistasis analysis revealed that these...... conditions bypassed the requirement for the meiotic activator Mei3. Since Mei3 is normally needed for inactivation of the meiosis-repressing protein kinase Pat1, this finding suggests that the strong Byr2 signal causes inactivation of Pat1 by an alternative mechanism. Consistent with this possibility, we...

  9. Identification of an allosteric pocket on human hsp70 reveals a mode of inhibition of this therapeutically important protein.

    Science.gov (United States)

    Rodina, Anna; Patel, Pallav D; Kang, Yanlong; Patel, Yogita; Baaklini, Imad; Wong, Michael J H; Taldone, Tony; Yan, Pengrong; Yang, Chenghua; Maharaj, Ronnie; Gozman, Alexander; Patel, Maulik R; Patel, Hardik J; Chirico, William; Erdjument-Bromage, Hediye; Talele, Tanaji T; Young, Jason C; Chiosis, Gabriela

    2013-12-19

    Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70. PMID:24239008

  10. Fluorescence-Based Codetection with Protein Markers Reveals Distinct Cellular Compartments for Altered MicroRNA Expression in Solid Tumors

    DEFF Research Database (Denmark)

    Sempere, Lorenzo F.; Preis, Meir; Yezefski, Todd;

    2010-01-01

    of altered miRNA expression in solid tumors, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed, paraffin-embedded tissue specimens. This ISH method was implemented to be compatible with routine......Purpose: High-throughput profiling experiments have linked altered expression of microRNAs (miRNA) to different types of cancer. Tumor tissues are a heterogeneous mixture of not only cancer cells, but also supportive and reactive tumor microenvironment elements. To clarify the clinical significance...... clinical immunohistochemical (IHC) assays to enable the detection of miRNAs and protein markers in the same tissue section for colocalization and functional studies. Experimental Design: We used this combined ISH/IHC assay to study a subset of cancer-associated miRNAs, including miRNAs frequently detected...

  11. Conserved synteny at the protein family level reveals genes underlying Shewanella species cold tolerance and predicts their novel phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Karpinets, Tatiana V.; Obraztsova, Anna; Wang, Yanbing; Schmoyer, Denise D.; Kora, Guruprasad; Park, Byung H.; Serres, Margrethe H.; Romine, Margaret F.; Land, Miriam L.; Kothe, Terence B.; Fredrickson, Jim K.; Nealson, Kenneth H.; Uberbacher, Edward

    2010-03-01

    Bacteria of the genus Shewanella can thrive in different environments and demonstrate significant variability in their metabolic and ecophysiological capabilities including cold and salt tolerance. Genomic characteristics underlying this variability across species are largely unknown. In this study we address the problem by a comparison of the physiological, metabolic and genomic characteristics of 19 sequenced Shewanella species. We have employed two novel approaches based on association of a phenotypic trait with the number of the trait-specific protein families (Pfam domains) and on the conservation of synteny (order in the genome) of the trait-related genes. Our first approach is top-down and involves experimental evaluation and quantification of the species’ cold tolerance followed by identification of the correlated Pfam domains and genes with a conserved synteny. The second, a bottom-up approach, predicts novel phenotypes of the species by calculating profiles of each Pfam domain among their genomes and following pair-wise correlation of the profiles and their network clustering. Using the first approach we find a link between cold and salt tolerance of the species and the presence in the genome of a Na+/H+ antiporter gene cluster. Other cold tolerance related genes includes peptidases, chemotaxis sensory transducer proteins, a cysteine exporter, and helicases. Using the bottom-up approach we found several novel phenotypes in the newly sequenced Shewanella species, including degradation of aromatic compounds by an aerobic hybrid pathway in S. woodyi, degradation of ethanolamine by S. benthica, and propanediol degradation by S. putrefaciens CN32 and S. sp. W3-18-1.

  12. Knockout studies reveal an important role of Plasmodium lipoic acid protein ligase A1 for asexual blood stage parasite survival.

    Directory of Open Access Journals (Sweden)

    Svenja Günther

    Full Text Available Lipoic acid (LA is a dithiol-containing cofactor that is essential for the function of alpha-keto acid dehydrogenase complexes. LA acts as a reversible acyl group acceptor and 'swinging arm' during acyl-coenzyme A formation. The cofactor is post-translationally attached to the acyl-transferase subunits of the multienzyme complexes through the action of octanoyl (lipoyl: N-octanoyl (lipoyl transferase (LipB or lipoic acid protein ligases (LplA. Remarkably, apicomplexan parasites possess LA biosynthesis as well as scavenging pathways and the two pathways are distributed between mitochondrion and a vestigial organelle, the apicoplast. The apicoplast-specific LipB is dispensable for parasite growth due to functional redundancy of the parasite's lipoic acid/octanoic acid ligases/transferases. In this study, we show that LplA1 plays a pivotal role during the development of the erythrocytic stages of the malaria parasite. Gene disruptions in the human malaria parasite P. falciparum consistently were unsuccessful while in the rodent malaria model parasite P. berghei the LplA1 gene locus was targeted by knock-in and knockout constructs. However, the LplA1((- mutant could not be cloned suggesting a critical role of LplA1 for asexual parasite growth in vitro and in vivo. These experimental genetics data suggest that lipoylation during expansion in red blood cells largely occurs through salvage from the host erythrocytes and subsequent ligation of LA to the target proteins of the malaria parasite.

  13. Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from Leishmania major reveals a unique protein fold.

    Science.gov (United States)

    Feliciano, Patricia R; Drennan, Catherine L; Nonato, M Cristina

    2016-08-30

    Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases. PMID:27528683

  14. Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

    OpenAIRE

    Ghodke, Harshad; Wang, Hong; Hsieh, Ching L.; Woldemeskel, Selamawit; Watkins, Simon C.; Rapić-Otrin, Vesna; Van Houten, Bennett

    2014-01-01

    UV damage in genomic DNA is identified by the human UV-damaged DNA-binding protein (UV-DDB). Recognition of DNA damage by UV-DDB serves to initiate global genomic nucleotide excision repair (NER) in humans. Recent work has revealed that UV-DDB dimerizes at sites of damage. This study demonstrates that prior to stable damage recognition, UV-DDB interrogates DNA for damage via a 3D diffusion mechanism coupled to the formation of multiple transient intermediates. Stable binding at sites of damag...

  15. Cryoelectron Microscopy Structure of the DNA-dependent Protein Kinase Catalytic Subunit (DNA-PKcs) at Subnanometer Resolution Reveals α-Helices and Insight into DNA Binding

    OpenAIRE

    Williams, Dewight R.; Lee, Kyung-Jong; Shi, Jian; Chen, David J.; Stewart, Phoebe L

    2008-01-01

    The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates the non-homologous end joining pathway for repair of double-stranded DNA breaks. Here we present a 7Å resolution structure of DNA-PKcs determined by cryoEM single particle reconstruction. This structure is composed of density rods throughout the molecule indicative of α-helices and reveals new structural features not observed in lower resolution EM structures. Docking of homology models into the DNA-PKcs structure demonst...

  16. Domestication process of the goat revealed by an analysis of the nearly complete mitochondrial protein-encoding genes.

    Directory of Open Access Journals (Sweden)

    Koh Nomura

    Full Text Available Goats (Capra hircus are one of the oldest domesticated species, and they are kept all over the world as an essential resource for meat, milk, and fiber. Although recent archeological and molecular biological studies suggested that they originated in West Asia, their domestication processes such as the timing of population expansion and the dynamics of their selection pressures are little known. With the aim of addressing these issues, the nearly complete mitochondrial protein-encoding genes were determined from East, Southeast, and South Asian populations. Our coalescent time estimations suggest that the timing of their major population expansions was in the Late Pleistocene and significantly predates the beginning of their domestication in the Neolithic era (≈10,000 years ago. The ω (ratio of non-synonymous rate/synonymous substitution rate for each lineage was also estimated. We found that the ω of the globally distributed haplogroup A which is inherited by more than 90% of goats examined, turned out to be extremely low, suggesting that they are under severe selection pressure probably due to their large population size. Conversely, the ω of the Asian-specific haplogroup B inherited by about 5% of goats was relatively high. Although recent molecular studies suggest that domestication of animals may tend to relax selective constraints, the opposite pattern observed in our goat mitochondrial genome data indicates the process of domestication is more complex than may be presently appreciated and cannot be explained only by a simple relaxation model.

  17. Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function During Nanoparticle-Induced Oxidative Stress

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Jicheng; Kodali, Vamsi K.; Gaffrey, Matthew J.; Guo, Jia; Chu, Rosalie K.; Camp, David G.; Smith, Richard D.; Thrall, Brian D.; Qian, Weijun

    2015-12-23

    Engineered nanoparticles (ENPs) are emerging functional materials increasingly utilized for commercial and medical applications. Due to the potential hazard effects of ENPs to human health, it is significant to assess and understand the underlying mechanisms of nanotoxicity. Herein, we investigate protein S-glutathionylation (SSG) as an underlying regulatory mechanism for ENP-induced oxidative stress in macrophages by applying a recently developed quantitative redox proteomics approach for site-specific measurements of SSG. Three high-volume production ENPs (SiO2, Fe3O4 and CoO) were selected as representative ENPs with low, moderate, and high reactive oxygen species (ROS) activity, respectively. Among these nanoparticles, we observe that CoO led to the most significant dose-dependent oxidative stress and increase of protein SSG modifications in macrophages. Our site-specific SSG changes highlighted a broad set of redox sensitive proteins and their specific Cys residues potentially implicated in stress response. Functional analysis revealed that the most significantly enriched functional categories for SSG-modified proteins were stress response, cellular structure change, and cell death or survival. Moreover, ENPs-induce oxidative stress levels (CoO > Fe3O4 > SiO2) were found to correlate well with the levels of impairment of macrophage phagocytic activity and the overall degrees of increases in SSG. RNA silencing knockdown experiment of glutaredoxin 1 (Grx1) also led to a decreased phagocytic activity in macrophages, which suggested a regulatory role of SSG in phagocytosis. Together, the results provided valuable insights of protein SSG as a potential regulatory mechanism in response to nanomaterial-induced oxidative stress and immunity dysfunction.

  18. Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration

    KAUST Repository

    Trujillo, Uldaeliz

    2013-02-28

    The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP

  19. Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

    Directory of Open Access Journals (Sweden)

    Uldaeliz Trujillo

    Full Text Available The polyunsaturated fatty acid (PUFA synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect and in structural stabilization of the multidomain protein (synergistic effect. While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of

  20. Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly.

    Directory of Open Access Journals (Sweden)

    Vlastimil Jirasko

    Full Text Available Non-structural protein 2 (NS2 plays an important role in hepatitis C virus (HCV assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.

  1. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    International Nuclear Information System (INIS)

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed

  2. An ultra-specific avian antibody to phosphorylated tau protein reveals a unique mechanism for phosphoepitope recognition.

    Science.gov (United States)

    Shih, Heather H; Tu, Chao; Cao, Wei; Klein, Anne; Ramsey, Renee; Fennell, Brian J; Lambert, Matthew; Ní Shúilleabháin, Deirdre; Autin, Bénédicte; Kouranova, Eugenia; Laxmanan, Sri; Braithwaite, Steven; Wu, Leeying; Ait-Zahra, Mostafa; Milici, Anthony J; Dumin, Jo Ann; LaVallie, Edward R; Arai, Maya; Corcoran, Christopher; Paulsen, Janet E; Gill, Davinder; Cunningham, Orla; Bard, Joel; Mosyak, Lydia; Finlay, William J J

    2012-12-28

    Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity. PMID:23148212

  3. Protein markers of cancer-associated fibroblasts and tumor-initiating cells reveal subpopulations in freshly isolated ovarian cancer ascites

    Directory of Open Access Journals (Sweden)

    Wintzell My

    2012-08-01

    Full Text Available Abstract Background In ovarian cancer, massive intraperitoneal dissemination is due to exfoliated tumor cells in ascites. Tumor-initiating cells (TICs or cancer stem cells and cells showing epithelial-mesenchymal-transition (EMT are particularly implicated. Spontaneous spherical cell aggregates are sometimes observed, but although similar to those formed by TICs in vitro, their significance is unclear. Methods Cells freshly isolated from malignant ascites were separated into sphere samples (S-type samples, n=9 and monolayer-forming single-cell suspensions (M-type, n=18. Using western blot, these were then compared for expression of protein markers of EMT, TIC, and of cancer-associated fibroblasts (CAFs. Results S-type cells differed significantly from M-type by expressing high levels of E-cadherin and no or little vimentin, integrin-β3 or stem cell transcription factor Oct-4A. By contrast, M-type samples were enriched for CD44, Oct-4A and for CAF markers. Independently of M- and S-type, there was a strong correlation between TIC markers Nanog and EpCAM. The CAF marker α-SMA correlated with clinical stage IV. This is the first report on CAF markers in malignant ascites and on SUMOylation of Oct-4A in ovarian cancer. Conclusions In addition to demonstrating potentially high levels of TICs in ascites, the results suggest that the S-type population is the less tumorigenic one. Nanoghigh/EpCAMhigh samples represent a TIC subset which may be either M- or S-type, and which is separate from the CD44high/Oct-4Ahigh subset observed only in M-type samples. This demonstrates a heterogeneity in TIC populations in vivo which has practical implications for TIC isolation based on cell sorting. The biological heterogeneity will need to be addressed in future therapeutical strategies.

  4. Structural and biochemical characterization of bacterial YpgQ protein reveals a metal-dependent nucleotide pyrophosphohydrolase.

    Science.gov (United States)

    Jeon, Ye Ji; Park, Sun Cheol; Song, Wan Seok; Kim, Ok-Hee; Oh, Byung-Chul; Yoon, Sung-Il

    2016-07-01

    The optimal balance of cellular nucleotides and the efficient elimination of non-canonical nucleotides are critical to avoiding erroneous mutation during DNA replication. One such mechanism involves the degradation of excessive or abnormal nucleotides by nucleotide-hydrolyzing enzymes. YpgQ contains the histidine-aspartate (HD) domain that is involved in the hydrolysis of nucleotides or nucleic acids, but the enzymatic activity and substrate specificity of YpgQ have never been characterized. Here, we unravel the catalytic activity and structural features of YpgQ to report the first Mn(2+)-dependent pyrophosphohydrolase that hydrolyzes (deoxy)ribonucleoside triphosphate [(d)NTP] to (deoxy)ribonucleoside monophosphate and pyrophosphate using the HD domain. YpgQ from Bacillus subtilis (bsYpgQ) displays a helical structure and assembles into a unique dimeric architecture that has not been observed in other HD domain-containing proteins. Each bsYpgQ monomer accommodates a metal ion and a nucleotide substrate in a cavity located between the N- and C-terminal lobes. The metal cofactor is coordinated by the canonical residues of the HD domain, namely, two histidine residues and two aspartate residues, and is positioned in close proximity to the β-phosphate group of the nucleotide, allowing us to propose a nucleophilic attack mechanism for the nucleotide hydrolysis reaction. YpgQ enzymes from other bacterial species also catalyze pyrophosphohydrolysis but exhibit different substrate specificity. Comparative structural and mutational studies demonstrated that residues outside the major substrate-binding site of bsYpgQ are responsible for the species-specific substrate preference. Taken together, our structural and biochemical analyses highlight the substrate-recognition mode and catalysis mechanism of YpgQ in pyrophosphohydrolysis. PMID:27062940

  5. Protein markers of cancer-associated fibroblasts and tumor-initiating cells reveal subpopulations in freshly isolated ovarian cancer ascites

    International Nuclear Information System (INIS)

    In ovarian cancer, massive intraperitoneal dissemination is due to exfoliated tumor cells in ascites. Tumor-initiating cells (TICs or cancer stem cells) and cells showing epithelial-mesenchymal-transition (EMT) are particularly implicated. Spontaneous spherical cell aggregates are sometimes observed, but although similar to those formed by TICs in vitro, their significance is unclear. Cells freshly isolated from malignant ascites were separated into sphere samples (S-type samples, n=9) and monolayer-forming single-cell suspensions (M-type, n=18). Using western blot, these were then compared for expression of protein markers of EMT, TIC, and of cancer-associated fibroblasts (CAFs). S-type cells differed significantly from M-type by expressing high levels of E-cadherin and no or little vimentin, integrin-β3 or stem cell transcription factor Oct-4A. By contrast, M-type samples were enriched for CD44, Oct-4A and for CAF markers. Independently of M- and S-type, there was a strong correlation between TIC markers Nanog and EpCAM. The CAF marker α-SMA correlated with clinical stage IV. This is the first report on CAF markers in malignant ascites and on SUMOylation of Oct-4A in ovarian cancer. In addition to demonstrating potentially high levels of TICs in ascites, the results suggest that the S-type population is the less tumorigenic one. Nanoghigh/EpCAMhigh samples represent a TIC subset which may be either M- or S-type, and which is separate from the CD44high/Oct-4Ahigh subset observed only in M-type samples. This demonstrates a heterogeneity in TIC populations in vivo which has practical implications for TIC isolation based on cell sorting. The biological heterogeneity will need to be addressed in future therapeutical strategies

  6. Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein-specific antibodies.

    Science.gov (United States)

    Höfling, Corinna; Morawski, Markus; Zeitschel, Ulrike; Zanier, Elisa R; Moschke, Katrin; Serdaroglu, Alperen; Canneva, Fabio; von Hörsten, Stephan; De Simoni, Maria-Grazia; Forloni, Gianluigi; Jäger, Carsten; Kremmer, Elisabeth; Roßner, Steffen; Lichtenthaler, Stefan F; Kuhn, Peer-Hendrik

    2016-10-01

    Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age-related and brain region-specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP-transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP-transgenic mouse and one APP-transgenic rat model. We observed remarkable differences in expression levels and brain region-specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP-transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals. PMID:27470171

  7. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death.

    Science.gov (United States)

    Qvit, Nir; Joshi, Amit U; Cunningham, Anna D; Ferreira, Julio C B; Mochly-Rosen, Daria

    2016-06-24

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme, has a non-catalytic (thus a non-canonical) role in inducing mitochondrial elimination under oxidative stress. We recently demonstrated that phosphorylation of GAPDH by δ protein kinase C (δPKC) inhibits this GAPDH-dependent mitochondrial elimination. δPKC phosphorylation of GAPDH correlates with increased cell injury following oxidative stress, suggesting that inhibiting GAPDH phosphorylation should decrease cell injury. Using rational design, we identified pseudo-GAPDH (ψGAPDH) peptide, an inhibitor of δPKC-mediated GAPDH phosphorylation that does not inhibit the phosphorylation of other δPKC substrates. Unexpectedly, ψGAPDH decreased mitochondrial elimination and increased cardiac damage in an animal model of heart attack. Either treatment with ψGAPDH or direct phosphorylation of GAPDH by δPKC decreased GAPDH tetramerization, which corresponded to reduced GAPDH glycolytic activity in vitro and ex vivo Taken together, our study identified the potential mechanism by which oxidative stress inhibits the protective GAPDH-mediated elimination of damaged mitochondria. Our study also identified a pharmacological tool, ψGAPDH peptide, with interesting properties. ψGAPDH peptide is an inhibitor of the interaction between δPKC and GAPDH and of the resulting phosphorylation of GAPDH by δPKC. ψGAPDH peptide is also an inhibitor of GAPDH oligomerization and thus an inhibitor of GAPDH glycolytic activity. Finally, we found that ψGAPDH peptide is an inhibitor of the elimination of damaged mitochondria. We discuss how this unique property of increasing cell damage following oxidative stress suggests a potential use for ψGAPDH peptide-based therapy. PMID:27129213

  8. Predicting the proteins of Angomonas deanei, Strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family.

    Directory of Open Access Journals (Sweden)

    Maria Cristina Machado Motta

    Full Text Available Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei and Strigomonas culicis (first known as Blastocrithidia culicis, respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine

  9. Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

    Science.gov (United States)

    Motta, Maria Cristina Machado; Martins, Allan Cezar de Azevedo; de Souza, Silvana Sant’Anna; Catta-Preta, Carolina Moura Costa; Silva, Rosane; Klein, Cecilia Coimbra; de Almeida, Luiz Gonzaga Paula; de Lima Cunha, Oberdan; Ciapina, Luciane Prioli; Brocchi, Marcelo; Colabardini, Ana Cristina; de Araujo Lima, Bruna; Machado, Carlos Renato; de Almeida Soares, Célia Maria; Probst, Christian Macagnan; de Menezes, Claudia Beatriz Afonso; Thompson, Claudia Elizabeth; Bartholomeu, Daniella Castanheira; Gradia, Daniela Fiori; Pavoni, Daniela Parada; Grisard, Edmundo C.; Fantinatti-Garboggini, Fabiana; Marchini, Fabricio Klerynton; Rodrigues-Luiz, Gabriela Flávia; Wagner, Glauber; Goldman, Gustavo Henrique; Fietto, Juliana Lopes Rangel; Elias, Maria Carolina; Goldman, Maria Helena S.; Sagot, Marie-France; Pereira, Maristela; Stoco, Patrícia H.; de Mendonça-Neto, Rondon Pessoa; Teixeira, Santuza Maria Ribeiro; Maciel, Talles Eduardo Ferreira; de Oliveira Mendes, Tiago Antônio; Ürményi, Turán P.; de Souza, Wanderley; Schenkman, Sergio; de Vasconcelos, Ana Tereza Ribeiro

    2013-01-01

    Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism

  10. Genomic characterization of the European sea bass Dicentrarchus labrax reveals the presence of a novel uncoupling protein (UCP gene family member in the teleost fish lineage

    Directory of Open Access Journals (Sweden)

    Tine Mbaye

    2012-05-01

    Full Text Available Abstract Background Uncoupling proteins (UCP are evolutionary conserved mitochondrial carriers that control energy metabolism and therefore play important roles in several physiological processes such as thermogenesis, regulation of reactive oxygen species (ROS, growth control, lipid metabolism and regulation of insulin secretion. Despite their importance in various physiological processes, their molecular function remains controversial. The evolution and phylogenetic distribution may assist to identify their general biological function and structure-function relationships. The exact number of uncoupling protein genes in the fish genome and their evolution is unresolved. Results Here we report the first characterisation of UCP gene family members in sea bass, Dicentrarchus labrax, and then retrace the evolution of the protein family in vertebrates. Four UCP genes that are shared by five other fish species were identified in sea bass genome. Phylogenetic reconstitution among vertebrate species and synteny analysis revealed that UCP1, UCP2 and UCP3 evolved from duplication events that occurred in the common ancestor of vertebrates, whereas the novel fourth UCP originated specifically in the teleost lineage. Functional divergence analysis among teleost species revealed specific amino acid positions that have been subjected to altered functional constraints after duplications. Conclusions This work provides the first unambiguous evidence for the presence of a fourth UCP gene in teleost fish genome and brings new insights into the evolutionary history of the gene family. Our results suggest functional divergence among paralogues which might result from long-term and differential selective pressures, and therefore, provide the indication that UCP genes may have diverse physiological functions in teleost fishes. Further experimental analysis of the critical amino acids identified here may provide valuable information on the physiological functions of

  11. Proteomic analysis of membrane microdomain-associated proteins in the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder reveals alterations in LAMP, STXBP1 and BASP1 protein expression.

    LENUS (Irish Health Repository)

    Behan, A T

    2009-06-01

    The dorsolateral prefrontal cortex (dlpfc) is strongly implicated in the pathogenesis of schizophrenia (SCZ) and bipolar disorder (BPD) and, within this region, abnormalities in glutamatergic neurotransmission and synaptic function have been described. Proteins associated with these functions are enriched in membrane microdomains (MM). In the current study, we used two complementary proteomic methods, two-dimensional difference gel electrophoresis and one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by reverse phase-liquid chromatography-tandem mass spectrometry (RP-LC-MS\\/MS) (gel separation liquid chromatography-tandem mass spectrometry (GeLC-MS\\/MS)) to assess protein expression in MM in pooled samples of dlpfc from SCZ, BPD and control cases (n=10 per group) from the Stanley Foundation Brain series. We identified 16 proteins altered in one\\/both disorders using proteomic methods. We selected three proteins with roles in synaptic function (syntaxin-binding protein 1 (STXBP1), brain abundant membrane-attached signal protein 1 (BASP1) and limbic system-associated membrane protein (LAMP)) for validation by western blotting. This revealed significantly increased expression of these proteins in SCZ (STXBP1 (24% difference; P<0.001), BASP1 (40% difference; P<0.05) and LAMP (22% difference; P<0.01)) and BPD (STXBP1 (31% difference; P<0.001), BASP1 (23% difference; P<0.01) and LAMP (20% difference; P<0.01)) in the Stanley brain series (n=20 per group). Further validation in dlpfc from the Harvard brain subseries (n=10 per group) confirmed increased protein expression in SCZ of STXBP1 (18% difference; P<0.0001), BASP1 (14% difference; P<0.0001) but not LAMP (20% difference; P=0.14). No significant differences in STXBP1, BASP1 or LAMP protein expression in BPD dlpfc were observed. This study, through proteomic assessments of MM in dlpfc and validation in two brain series, strongly implicates LAMP, STXBP1 and BASP1 in SCZ and supports

  12. The short-lived signaling state of the photoactive yellow protein photoreceptor revealed by combined structural probes.

    Science.gov (United States)

    Ramachandran, Pradeep L; Lovett, Janet E; Carl, Patrick J; Cammarata, Marco; Lee, Jae Hyuk; Jung, Yang Ouk; Ihee, Hyotcherl; Timmel, Christiane R; van Thor, Jasper J

    2011-06-22

    The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump-probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 Å upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from (1)H-(15)N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump-probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the

  13. Genome-wide analysis on Chlamydomonas reinhardtii reveals the impact of hydrogen peroxide on protein stress responses and overlap with other stress transcriptomes.

    Science.gov (United States)

    Blaby, Ian K; Blaby-Haas, Crysten E; Pérez-Pérez, María Esther; Schmollinger, Stefan; Fitz-Gibbon, Sorel; Lemaire, Stéphane D; Merchant, Sabeeha S

    2015-12-01

    Reactive oxygen species (ROS) are produced by and have the potential to be damaging to all aerobic organisms. In photosynthetic organisms, they are an unavoidable byproduct of electron transfer in both the chloroplast and mitochondrion. Here, we employ the reference unicellular green alga Chlamydomonas reinhardtii to identify the effect of H2O2 on gene expression by monitoring the changes in the transcriptome in a time-course experiment. Comparison of transcriptomes from cells sampled immediately prior to the addition of H2O2 and 0.5 and 1 h subsequently revealed 1278 differentially abundant transcripts. Of those transcripts that increase in abundance, many encode proteins involved in ROS detoxification, protein degradation and stress responses, whereas among those that decrease are transcripts encoding proteins involved in photosynthesis and central carbon metabolism. In addition to these transcriptomic adjustments, we observe that addition of H2O2 is followed by an accumulation and oxidation of the total intracellular glutathione pool, and a decrease in photosynthetic O2 output. Additionally, we analyze our transcriptomes in the context of changes in transcript abundance in response to singlet O2 (O2*), and relate our H2O2 -induced transcripts to a diurnal transcriptome, where we demonstrate enrichments of H2O2 -induced transcripts early in the light phase, late in the light phase and 2 h prior to light. On this basis several genes that are highlighted in this work may be involved in previously undiscovered stress remediation pathways or acclimation responses. PMID:26473430

  14. Solution Binding and Structural Analyses Reveal Potential Multidrug Resistance Functions for SAV2435 and CTR107 and Other GyrI-like Proteins.

    Science.gov (United States)

    Moreno, Andrew; Froehlig, John R; Bachas, Sharrol; Gunio, Drew; Alexander, Teressa; Vanya, Aaron; Wade, Herschel

    2016-08-30

    Multidrug resistance (MDR) refers to the acquired ability of cells to tolerate a broad range of toxic compounds. One mechanism cells employ is to increase the level of expression of efflux pumps for the expulsion of xenobiotics. A key feature uniting efflux-related mechanisms is multidrug (MD) recognition, either by efflux pumps themselves or by their transcriptional regulators. However, models describing MD binding by MDR effectors are incomplete, underscoring the importance of studies focused on the recognition elements and key motifs that dictate polyspecific binding. One such motif is the GyrI-like domain, which is found in several MDR proteins and is postulated to have been adapted for small-molecule binding and signaling. Here we report the solution binding properties and crystal structures of two proteins containing GyrI-like domains, SAV2435 and CTR107, bound to various ligands. Furthermore, we provide a comparison with deposited crystal structures of GyrI-like proteins, revealing key features of GyrI-like domains that not only support polyspecific binding but also are conserved among GyrI-like domains. Together, our studies suggest that GyrI-like domains perform evolutionarily conserved functions connected to multidrug binding and highlight the utility of these types of studies for elucidating mechanisms of MDR. PMID:27505298

  15. The calcium-dependent protein kinase 3 of toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis.

    Directory of Open Access Journals (Sweden)

    Moritz Treeck

    2014-06-01

    Full Text Available Calcium-dependent protein kinases (CDPKs are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC. This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1, as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

  16. Structural characterization of S100A15 reveals a novel zinc coordination site among S100 proteins and altered surface chemistry with functional implications for receptor binding

    Directory of Open Access Journals (Sweden)

    Murray Jill I

    2012-07-01

    Full Text Available Abstract Background S100 proteins are a family of small, EF-hand containing calcium-binding signaling proteins that are implicated in many cancers. While the majority of human S100 proteins share 25-65% sequence similarity, S100A7 and its recently identified paralog, S100A15, display 93% sequence identity. Intriguingly, however, S100A7 and S100A15 serve distinct roles in inflammatory skin disease; S100A7 signals through the receptor for advanced glycation products (RAGE in a zinc-dependent manner, while S100A15 signals through a yet unidentified G-protein coupled receptor in a zinc-independent manner. Of the seven divergent residues that differentiate S100A7 and S100A15, four cluster in a zinc-binding region and the remaining three localize to a predicted receptor-binding surface. Results To investigate the structural and functional consequences of these divergent clusters, we report the X-ray crystal structures of S100A15 and S100A7D24G, a hybrid variant where the zinc ligand Asp24 of S100A7 has been substituted with the glycine of S100A15, to 1.7 Å and 1.6 Å resolution, respectively. Remarkably, despite replacement of the Asp ligand, zinc binding is retained at the S100A15 dimer interface with distorted tetrahedral geometry and a chloride ion serving as an exogenous fourth ligand. Zinc binding was confirmed using anomalous difference maps and solution binding studies that revealed similar affinities of zinc for S100A15 and S100A7. Additionally, the predicted receptor-binding surface on S100A7 is substantially more basic in S100A15 without incurring structural rearrangement. Conclusions Here we demonstrate that S100A15 retains the ability to coordinate zinc through incorporation of an exogenous ligand resulting in a unique zinc-binding site among S100 proteins. The altered surface chemistry between S100A7 and S100A15 that localizes to the predicted receptor binding site is likely responsible for the differential recognition of distinct

  17. Loss of mitogen-activated protein kinase kinase kinase 4 (MAP3K4 reveals a requirement for MAPK signalling in mouse sex determination.

    Directory of Open Access Journals (Sweden)

    Debora Bogani

    2009-09-01

    Full Text Available Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY gonad, sex-determining region of the Y (SRY protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4, a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas. These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and

  18. Structure of the retinoblastoma protein bound to adenovirus E1A reveals the molecular basis for viral oncoprotein inactivation of a tumor suppressor

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin; Marmorstein, Ronen (UPENN)

    2008-04-02

    The adenovirus (Ad) E1A (Ad-E1A) oncoprotein mediates cell transformation, in part, by displacing E2F transcription factors from the retinoblastoma protein (pRb) tumor suppressor. In this study we determined the crystal structure of the pRb pocket domain in complex with conserved region 1 (CR1) of Ad5-E1A. The structure and accompanying biochemical studies reveal that E1A-CR1 binds at the interface of the A and B cyclin folds of the pRb pocket domain, and that both E1A-CR1 and the E2F transactivation domain use similar conserved nonpolar residues to engage overlapping sites on pRb, implicating a novel molecular mechanism for pRb inactivation by a viral oncoprotein.

  19. Stamen abscission zone transcriptome profiling reveals new candidates for abscission control: enhanced retention of floral organs in transgenic plants overexpressing Arabidopsis ZINC FINGER PROTEIN2.

    Science.gov (United States)

    Cai, Suqin; Lashbrook, Coralie C

    2008-03-01

    Organ detachment requires cell separation within abscission zones (AZs). Physiological studies have established that ethylene and auxin contribute to cell separation control. Genetic analyses of abscission mutants have defined ethylene-independent detachment regulators. Functional genomic strategies leading to global understandings of abscission have awaited methods for isolating AZ cells of low abundance and very small size. Here, we couple laser capture microdissection of Arabidopsis thaliana stamen AZs and GeneChip profiling to reveal the AZ transcriptome responding to a developmental shedding cue. Analyses focus on 551 AZ genes (AZ(551)) regulated at the highest statistical significance (P Gene Ontology Consortium functional categories for cell wall modifying proteins, extracellular regulators, and nuclear-residing transcription factors. Promoter-beta-glucuronidase expression of one transcription factor candidate, ZINC FINGER PROTEIN2 (AtZFP2), was elevated in stamen, petal, and sepal AZs. Flower parts of transgenic lines overexpressing AtZFP2 exhibited asynchronous and delayed abscission. Abscission defects were accompanied by altered floral morphology limiting pollination and fertility. Hand-pollination restored transgenic fruit development but not the rapid abscission seen in wild-type plants, demonstrating that pollination does not assure normal rates of detachment. In wild-type stamen AZs, AtZFP2 is significantly up-regulated postanthesis. Phenotype data from transgene overexpression studies suggest that AtZFP2 participates in processes that directly or indirectly influence organ shed. PMID:18192438

  20. Direct comparison of mice null for liver or intestinal fatty acid-binding proteins reveals highly divergent phenotypic responses to high fat feeding.

    Science.gov (United States)

    Gajda, Angela M; Zhou, Yin Xiu; Agellon, Luis B; Fried, Susan K; Kodukula, Sarala; Fortson, Walter; Patel, Khamoshi; Storch, Judith

    2013-10-18

    The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal FABP (IFABP; FABP2) and liver FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally coexpressed proteins, which both bind long chain fatty acids (FA), are functionally distinct. Here, we directly compared IFABP(-/-) and LFABP(-/-) mice fed high fat diets containing long chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP(-/-) mice, whereas LFABP(-/-) mice had reduced monoacylglycerol incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis; LFABP(-/-) mice fed high fat diets became obese relative to WT, whereas IFABP(-/-) mice displayed an opposite, lean phenotype. Fuel utilization followed adiposity, with LFABP(-/-) mice preferentially utilizing lipids, and IFABP(-/-) mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamine were elevated in LFABP(-/-), perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP(-/-) mice, result in divergent downstream effects at the systemic level. PMID:23990461

  1. Magic Angle Spinning NMR Reveals Sequence-Dependent Structural Plasticity, Dynamics, and the Spacer Peptide 1 Conformation in HIV-1 Capsid Protein Assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yun; Hou, Guangjin; Suiter, Christopher L.; Ahn, Jinwoo; Byeon, In-Ja L.; Lipton, Andrew S.; Burton, Sarah D.; Hung, Ivan; Gorkov, Peter L.; Gan, Zhehong; Brey, William W.; Rice, David M.; Gronenborn, Angela M.; Polenova, Tatyana E.

    2013-11-27

    Maturation of HIV-1 virus into an infectious virion requires cleavage of the Gag polyprotein into its constituent domains and formation of a conical capsid core that encloses viral RNA and a small complement of proteins for replication. The final step of this process is the cleavage of the SP1 peptide from the CA-SP1 maturation intermediate, which triggers the condensation of the CA protein into a conical capsid. The mechanism of this step, including the conformation of the SP1 peptide in CA-SP1, is under intense debate. In this report, we examine the tubular assemblies of CA and the CA-SP1 maturation intermediate using Magic Angle Spinning NMR spectroscopy. At the magnetic fields of 19.9 T and above, tubular CA and CA-SP1 assemblies yield outstanding-quality 2D and 3D MAS NMR spectra, which are amenable to resonance assignments and detailed structural characterization. Dipolar- and scalar-based correlation experiments unequivocally indicate that SP1 peptide is in a random coil conformation and mobile in the assembled CA-SP1. Analysis of two sequence variants reveals that remarkably, the conformation of SP1 tail, of the functionally important CypA loop, and of the loop preceding helix 8 are sequence dependent and modulated by the residue variations at distal sites. These findings challenge the role of SP1 as a conformational switch in the maturation process and establish sequence-dependent conformational plasticity in CA.

  2. The structure of Aquifex aeolicus ribosomal protein S8 reveals a unique subdomain that contributes to an extremely tight association with 16S rRNA.

    Science.gov (United States)

    Menichelli, Elena; Edgcomb, Stephen P; Recht, Michael I; Williamson, James R

    2012-01-20

    The assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. The ribosomal protein S8 from Aquifex aeolicus (AS8) is unique in that there is a 41-residue insertion in the consensus S8 sequence. In addition, AS8 exhibits an unusually high affinity for the 16S ribosomal RNA, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than the equivalent interaction from Escherichia coli. Deletion analysis demonstrated that binding to the minimal site on helix 21 occurred at the same nanomolar affinity found for other bacterial species. The additional affinity required the presence of a three-helix junction between helices 20, 21, and 22. The crystal structure of AS8 was solved, revealing the helix-loop-helix geometry of the unique AS8 insertion region, while the core of the molecule is conserved with known S8 structures. The AS8 structure was modeled onto the structure of the 30S ribosomal subunit from E. coli, suggesting the possibility that the unique subdomain provides additional backbone and side-chain contacts between the protein and an unpaired base within the three-way junction of helices 20, 21, and 22. Point mutations in the protein insertion subdomain resulted in a significantly reduced RNA binding affinity with respect to wild-type AS8. These results indicate that the AS8-specific subdomain provides additional interactions with the three-way junction that contribute to the extremely tight binding to ribosomal RNA. PMID:22079365

  3. Systematic microRNAome profiling reveals the roles of microRNAs in milk protein metabolism and quality: insights on low-quality forage utilization.

    Science.gov (United States)

    Wang, Diming; Liang, Guanxiang; Wang, Bing; Sun, Huizeng; Liu, Jianxin; Guan, Le Luo

    2016-01-01

    In this study, we investigated the molecular regulatory mechanisms of milk protein production in dairy cows by studying the miRNAomes of five key metabolic tissues involved in protein synthesis and metabolism from dairy cows fed high- and low-quality diets. In total, 340, 338, 337, 330, and 328 miRNAs were expressed in the rumen, duodenum, jejunum, liver, and mammary gland tissues, respectively. Some miRNAs were highly correlated with feed and nitrogen efficiency, with target genes involved in transportation and phosphorylation of amino acid (AA). Additionally, low-quality forage diets (corn stover and rice straw) influenced the expression of feed and nitrogen efficiency-associated miRNAs such as miR-99b in rumen, miR-2336 in duodenum, miR-652 in jejunum, miR-1 in liver, and miR-181a in mammary gland. Ruminal miR-21-3p and liver miR-2285f were predicted to regulate AA transportation by targeting ATP1A2 and SLC7A8, respectively. Furthermore, bovine-specific miRNAs regulated the proliferation and morphology of rumen epithelium, as well as the metabolism of liver lipids and branched-chain AAs, revealing bovine-specific mechanisms. Our results suggest that miRNAs expressed in these five tissues play roles in regulating transportation of AA for downstream milk production, which is an important mechanism that may be associated with low milk protein under low-quality forage feed. PMID:26884323

  4. Secreted protein gene derived-single nucleotide polymorphisms (SP-SNPs) reveal population diversity and differentiation of Puccinia striiformis f. sp. tritici in the United States.

    Science.gov (United States)

    Xia, Chongjing; Wan, Anmin; Wang, Meinan; Jiwan, Derick A; See, Deven R; Chen, Xianming

    2016-05-01

    Single nucleotide polymorphism (SNP) is a powerful molecular marker technique that has been widely used in population genetics and molecular mapping studies for various organisms. However, the technique has not been used for studying Puccinia striiformis f. sp. tritici (Pst), the wheat stripe rust pathogen. In this study, we developed over a hundred secreted protein gene-derived SNP (SP-SNP) markers and used 92 markers to study the population structure of Pst. From 352 isolates collected in the United States, we identified 242 multi-locus genotypes. The SP-SNP genotypes had a moderate, but significant correlation with the virulence phenotype data. Clustering of the multi-locus genotypes was consistent by various analyses, revealing distinct genetic groups. Analysis of molecular variance detected significant differences between the eastern and western US Pst populations. High heterozygosity was found in the US population with significant differences identified among epidemiological regions. Analysis of population differentiation revealed that populations between the eastern and western US were highly differentiated while moderate differentiation was found in populations within the western or eastern US. Isolates from the western US were more diverse than isolates from the eastern US. The information is useful for guiding the disease management in different epidemiological regions. PMID:27109369

  5. Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril.

    Science.gov (United States)

    Weerkamp, A H; Handley, P S; Baars, A; Slot, J W

    1986-01-01

    The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease

  6. Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dong-Hwa; Ha, Ji-Hyang [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Kim, Yul [Department of Bio and Brain Engineering, KAIST, Daejeon 305-701 (Korea, Republic of); Bae, Kwang-Hee [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Park, Jae-Yong [Department of Physiology, Institute of Health Science, School of Medicine, Gyeongsang National University, Jinju, Gyeongnam 660-751 (Korea, Republic of); Choi, Wan Sung [Department of Anatomy and Neurobiology, Institute of Health Science, School of Medicine, Gyeongsang National University, Jinju, Gyeongnam 660-751 (Korea, Republic of); Yoon, Ho Sup [Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637511 (Singapore); Park, Sung Goo; Park, Byoung Chul [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Yi, Gwan-Su, E-mail: gsyi@kaist.ac.kr [Department of Bio and Brain Engineering, KAIST, Daejeon 305-701 (Korea, Republic of); Chi, Seung-Wook, E-mail: swchi@kribb.re.kr [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of)

    2011-05-20

    Highlights: {yields} Identification of a conserved BH3 motif in C-terminal coiled coil region of nCLU. {yields} The nCLU BH3 domain binds to BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. {yields} A conserved binding mechanism of nCLU BH3 and the other pro-apoptotic BH3 peptides with Bcl-X{sub L}. {yields} The absolutely conserved Leu323 and Asp328 of nCLU BH3 domain are critical for binding to Bcl-X{sub L.} {yields} Molecular understanding of the pro-apoptotic function of nCLU as a novel BH3-only protein. -- Abstract: Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. A structural model of the Bcl-X{sub L}/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X{sub L}/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X{sub L}. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.

  7. Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

    International Nuclear Information System (INIS)

    Highlights: → Identification of a conserved BH3 motif in C-terminal coiled coil region of nCLU. → The nCLU BH3 domain binds to BH3 peptide-binding grooves in both Bcl-XL and Bcl-2. → A conserved binding mechanism of nCLU BH3 and the other pro-apoptotic BH3 peptides with Bcl-XL. → The absolutely conserved Leu323 and Asp328 of nCLU BH3 domain are critical for binding to Bcl-XL. → Molecular understanding of the pro-apoptotic function of nCLU as a novel BH3-only protein. -- Abstract: Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-XL and Bcl-2. A structural model of the Bcl-XL/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-XL/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-XL. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.

  8. Gene expression profiling in the stress control brain region hypothalamic paraventricular nucleus reveals a novel gene network including Amyloid beta Precursor Protein

    Directory of Open Access Journals (Sweden)

    Deussing Jan M

    2010-10-01

    Full Text Available Abstract Background The pivotal role of stress in the precipitation of psychiatric diseases such as depression is generally accepted. This study aims at the identification of genes that are directly or indirectly responding to stress. Inbred mouse strains that had been evidenced to differ in their stress response as well as in their response to antidepressant treatment were chosen for RNA profiling after stress exposure. Gene expression and regulation was determined by microarray analyses and further evaluated by bioinformatics tools including pathway and cluster analyses. Results Forced swimming as acute stressor was applied to C57BL/6J and DBA/2J mice and resulted in sets of regulated genes in the paraventricular nucleus of the hypothalamus (PVN, 4 h or 8 h after stress. Although the expression changes between the mouse strains were quite different, they unfolded in phases over time in both strains. Our search for connections between the regulated genes resulted in potential novel signalling pathways in stress. In particular, Guanine nucleotide binding protein, alpha inhibiting 2 (GNAi2 and Amyloid β (A4 precursor protein (APP were detected as stress-regulated genes, and together with other genes, seem to be integrated into stress-responsive pathways and gene networks in the PVN. Conclusions This search for stress-regulated genes in the PVN revealed its impact on interesting genes (GNAi2 and APP and a novel gene network. In particular the expression of APP in the PVN that is governing stress hormone balance, is of great interest. The reported neuroprotective role of this molecule in the CNS supports the idea that a short acute stress can elicit positive adaptational effects in the brain.

  9. Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

    Science.gov (United States)

    Ghodke, Harshad; Wang, Hong; Hsieh, Ching L.; Woldemeskel, Selamawit; Watkins, Simon C.; Rapić-Otrin, Vesna; Van Houten, Bennett

    2014-01-01

    How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3–0.8, 8.1, and 113–126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism. PMID:24760829

  10. Direct Comparison of Mice Null for Liver or Intestinal Fatty Acid-binding Proteins Reveals Highly Divergent Phenotypic Responses to High Fat Feeding*

    Science.gov (United States)

    Gajda, Angela M.; Zhou, Yin Xiu; Agellon, Luis B.; Fried, Susan K.; Kodukula, Sarala; Fortson, Walter; Patel, Khamoshi; Storch, Judith

    2013-01-01

    The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal FABP (IFABP; FABP2) and liver FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally coexpressed proteins, which both bind long chain fatty acids (FA), are functionally distinct. Here, we directly compared IFABP−/− and LFABP−/− mice fed high fat diets containing long chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP−/− mice, whereas LFABP−/− mice had reduced monoacylglycerol incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis; LFABP−/− mice fed high fat diets became obese relative to WT, whereas IFABP−/− mice displayed an opposite, lean phenotype. Fuel utilization followed adiposity, with LFABP−/− mice preferentially utilizing lipids, and IFABP−/− mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamine were elevated in LFABP−/−, perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP−/− mice, result in divergent downstream effects at the systemic level. PMID:23990461

  11. The Structure of Plasmodium falciparum Blood-Stage 6-Cys Protein Pf41 Reveals an Unexpected Intra-Domain Insertion Required for Pf12 Coordination.

    Directory of Open Access Journals (Sweden)

    Michelle L Parker

    Full Text Available Plasmodium falciparum is an apicomplexan parasite and the etiological agent of severe human malaria. The complex P. falciparum life cycle is supported by a diverse repertoire of surface proteins including the family of 6-Cys s48/45 antigens. Of these, Pf41 is localized to the surface of the blood-stage merozoite through its interaction with the glycophosphatidylinositol-anchored Pf12. Our recent structural characterization of Pf12 revealed two juxtaposed 6-Cys domains (D1 and D2. Pf41, however, contains an additional segment of 120 residues predicted to form a large spacer separating its two 6-Cys domains. To gain insight into the assembly mechanism and overall architecture of the Pf12-Pf41 complex, we first determined the 2.45 Å resolution crystal structure of Pf41 using zinc single-wavelength anomalous dispersion. Structural analysis revealed an unexpected domain organization where the Pf41 6-Cys domains are, in fact, intimately associated and the additional residues instead map predominately to an inserted domain-like region (ID located between two β-strands in D1. Notably, the ID is largely proteolyzed in the final structure suggesting inherent flexibility. To assess the contribution of the ID to complex formation, we engineered a form of Pf41 where the ID was replaced by a short glycine-serine linker and showed by isothermal titration calorimetry that binding to Pf12 was abrogated. Finally, protease protection assays showed that the proteolytic susceptibility of the ID was significantly reduced in the complex, consistent with the Pf41 ID directly engaging Pf12. Collectively, these data establish the architectural organization of Pf41 and define an essential role for the Pf41 ID in promoting assembly of the Pf12-Pf41 heterodimeric complex.

  12. Hepatitis B virus X protein (HBx)-induced abnormalities of nucleic acid metabolism revealed by 1H-NMR-based metabonomics

    Science.gov (United States)

    Dan Yue; Zhang, Yuwei; Cheng, Liuliu; Ma, Jinhu; Xi, Yufeng; Yang, Liping; Su, Chao; Shao, Bin; Huang, Anliang; Xiang, Rong; Cheng, Ping

    2016-01-01

    Hepatitis B virus X protein (HBx) plays an important role in HBV-related hepatocarcinogenesis; however, mechanisms underlying HBx-mediated carcinogenesis remain unclear. In this study, an NMR-based metabolomics approach was applied to systematically investigate the effects of HBx on cell metabolism. EdU incorporation assay was conducted to examine the effects of HBx on DNA synthesis, an important feature of nucleic acid metabolism. The results revealed that HBx disrupted metabolism of glucose, lipids, and amino acids, especially nucleic acids. To understand the potential mechanism of HBx-induced abnormalities of nucleic acid metabolism, gene expression profiles of HepG2 cells expressing HBx were investigated. The results showed that 29 genes involved in DNA damage and DNA repair were differentially expressed in HBx-expressing HepG2 cells. HBx-induced DNA damage was further demonstrated by karyotyping, comet assay, Western blotting, immunofluorescence and immunohistochemistry analyses. Many studies have previously reported that DNA damage can induce abnormalities of nucleic acid metabolism. Thus, our results implied that HBx initially induces DNA damage, and then disrupts nucleic acid metabolism, which in turn blocks DNA repair and induces the occurrence of hepatocellular carcinoma (HCC). These findings further contribute to our understanding of the occurrence of HCC. PMID:27075403

  13. Transcriptome analysis of the couch potato (CPO) protein reveals an expression pattern associated with early development in the salmon louse Caligus rogercresseyi.

    Science.gov (United States)

    Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Nuñez-Acuña, Gustavo; Chávez-Mardones, Jacqueline; Maldonado-Aguayo, Waleska

    2014-02-15

    The couch potato (CPO) protein is a key biomolecule involved in regulating diapause through the RNA-binding process of the peripheral and central nervous systems in insects and also recently discovered in a few crustacean species. As such, ectoparasitic copepods are interesting model species that have no evidence of developmental arrest. The present study is the first to report on the cloning of a putative CPO gene from the salmon louse Caligus rogercresseyi (CrCPO), as identified by high-throughput transcriptome sequencing. In addition, the transcription expression in larvae and adults was evaluated using quantitative real-time PCR. The CrCPO cDNA sequence showed 3261 base pairs (bp), consisting of 713bp of 5' UTR, 1741bp of 3' UTR, and an open reading frame of 807bp encoding for 268 amino acids. The highly conserved RNA binding regions RNP2 (LFVSGL) and RNP1 (SPVGFVTF), as well the dimerization site (LEF), were also found. Furthermore, eight single nucleotide polymorphisms located in the untranslated regions and one located in the coding region were detected. Gene transcription analysis revealed that CrCPO has ubiquitous expression across larval stages and in adult individuals, with the highest expression from nauplius to copepodid stages. The present study suggests a putative biological function of CrCPO associated with the development of the nervous system in salmon lice and contributes molecular evidence for candidate genes related to host-parasite interactions. PMID:24342663

  14. Royal jelly-like protein localization reveals differences in hypopharyngeal glands buildup and conserved expression pattern in brains of bumblebees and honeybees

    Directory of Open Access Journals (Sweden)

    Štefan Albert

    2014-03-01

    Full Text Available Royal jelly proteins (MRJPs of the honeybee bear several open questions. One of them is their expression in tissues other than the hypopharyngeal glands (HGs, the site of royal jelly production. The sole MRJP-like gene of the bumblebee, Bombus terrestris (BtRJPL, represents a pre-diversification stage of the MRJP gene evolution in bees. Here we investigate the expression of BtRJPL in the HGs and the brain of bumblebees. Comparison of the HGs of bumblebees and honeybees revealed striking differences in their morphology with respect to sex- and caste-specific appearance, number of cells per acinus, and filamentous actin (F-actin rings. At the cellular level, we found a temporary F-actin-covered meshwork in the secretory cells, which suggests a role for actin in the biogenesis of the end apparatus in HGs. Using immunohistochemical localization, we show that BtRJPL is expressed in the bumblebee brain, predominantly in the Kenyon cells of the mushroom bodies, the site of sensory integration in insects, and in the optic lobes. Our data suggest that a dual gland-brain function preceded the multiplication of MRJPs in the honeybee lineage. In the course of the honeybee evolution, HGs dramatically changed their morphology in order to serve a food-producing function.

  15. Not All Inner Ears are the Same: Otolith Matrix Proteins in the Inner Ear of Sub-Adult Cichlid Fish, Oreochromis Mossambicus, Reveal Insights Into the Biomineralization Process.

    Science.gov (United States)

    Weigele, Jochen; Franz-Odendaal, Tamara A; Hilbig, Reinhard

    2016-02-01

    The fish ear stones (otoliths) consist mainly of calcium carbonate and have lower amounts of a proteinous matrix. This matrix consists of macromolecules, which directly control the biomineralization process. We analyzed the composition of this proteinous matrix by mass spectrometry in a shotgun approach. For this purpose, an enhanced protein purification technique was developed that excludes any potential contamination of proteins from body fluids. Using this method we identified eight proteins in the inner ear of Oreochromis mossambicus. These include the common otolith matrix proteins (OMP-1, otolin-1, neuroserpin, SPARC and otoconin), and three proteins (alpha tectorin, otogelin and transferrin) not previously localized to the otoliths. Moreover, we were able to exclude the occurrence of two matrix proteins (starmaker and pre-cerebellin-like protein) known from other fish species. In further analyses, we show that the absence of the OMP starmaker corresponds to calcitic otoliths and that pre-cerebellin-like protein is not present at any stage during the development of the otoliths of the inner ear. This study shows O. mossambicus does not have all of the known otolith proteins indicating that the matrix proteins in the inner ear of fish are not the same across species. Further functional studies of the novel proteins we identified during otolith development are required. PMID:26559654

  16. Comparative proteomics reveals highly and differentially expressed proteins in field-collected and laboratory-cultured blooming cells of the diatom Skeletonema costatum.

    Science.gov (United States)

    Zhang, Hao; Wang, Da-Zhi; Xie, Zhang-Xian; Zhang, Shu-Fei; Wang, Ming-Hua; Lin, Lin

    2015-10-01

    Diatoms are a major phytoplankton group causing extensive blooms in the ocean. However, little is known about the intracellular biological processes occurring during the blooming period. This study compared the protein profiles of field-collected and laboratory-cultured blooming cells of Skeletonema costatum, and identified highly and differentially expressed proteins using the shotgun proteomic approach. A total of 1372 proteins were confidently identified with two or more peptides. Among them, 222 and 311 proteins were unique to the laboratory and field samples respectively. Proteins involved in photosynthesis, translation, nucleosome assembly, carbohydrate and energy metabolism dominated the protein profiles in both samples. However, different features of specific proteins were also found: proteins participated in light harvesting, photosynthetic pigment biosynthesis, photoprotection, cell division and redox homeostasis were highly detected in the field sample, whereas proteins involved in translation, amino acid and protein metabolic processes, and nitrogen and carbon assimilation presented high detection rates in the laboratory sample. ATP synthase cf1 subunit beta and light harvest complex protein were the most abundant protein in the laboratory and field samples respectively. These results indicated that S. costatum had evolved adaptive mechanisms to the changing environment, and integrating field and laboratory proteomic data should provide comprehensive understanding of bloom mechanisms. PMID:26014042

  17. Investigation of Fasciculation and Elongation Protein ζ-1 (FEZ1 in Peripheral Blood Reveals Differences in Gene Expression in Patients with Schizophrenia

    Directory of Open Access Journals (Sweden)

    Vachev T.I.

    2015-06-01

    Full Text Available Schizophrenia (SZ is a chronic neuropsychiatric disorder characterized by affective, neuromorphological and cognitive impairment, deteriorated social functioning and psychosis with underlying molecular abnormalities, including gene expression changes. Observations have suggested that fasciculation and elongation protein ζ-1 (FEZ1 may be implicated in the pathogenesis of schizophrenia. Nevertheless, our current knowledge of the expression of FEZ1 in peripheral blood of schizophrenia patients remains unclear. The purpose of this study was to identify the characteristic gene expression patterns of FEZ1 in peripheral blood samples from schizophrenia patients. We performed quantitative reverse-transcriptase (qRT-PCR analysis using peripheral blood from drug-free schizophrenia patients (n = 29 and age and gender-matched general population controls (n = 24. For the identification of FEZ1 gene expression patterns, we applied a comparative threshold cycle (CT method. A statistically significant difference of FEZ1 mRNA level was revealed in schizophrenia subjects compared to healthy controls (p = 0.0034. To the best of our knowledge, this study is the first describing a down-regulation of FEZ1 gene expression in peripheral blood of patients with schizophrenia. Our results suggested a possible functional role of FEZ1 in the pathogenesis of schizophrenia and confirmed the utility of peripheral blood samples for molecular profiling of psychiatric disorders including schizophrenia. The current study describes FEZ1 gene expression changes in peripheral blood of patients with schizophrenia with significantly down-regulation of FEZ1 mRNA. Thus, our results provide support for a model of SZ pathogenesis that includes the effects of FEZ1 expression.

  18. Crystal structure of enoyl-acyl carrier protein reductase (FabK) from Streptococcus pneumoniae reveals the binding mode of an inhibitor.

    Science.gov (United States)

    Saito, Jun; Yamada, Mototsugu; Watanabe, Takashi; Iida, Maiko; Kitagawa, Hideo; Takahata, Sho; Ozawa, Tomohiro; Takeuchi, Yasuo; Ohsawa, Fukuichi

    2008-04-01

    Enoyl-acyl carrier protein (ACP) reductases are critical for bacterial type II fatty acid biosynthesis and thus are attractive targets for developing novel antibiotics. We determined the crystal structure of enoyl-ACP reductase (FabK) from Streptococcus pneumoniae at 1.7 A resolution. There was one dimer per asymmetric unit. Each subunit formed a triose phosphate isomerase (TIM) barrel structure, and flavin mononucleotide (FMN) was bound as a cofactor in the active site. The overall structure was similar to the enoyl-ACP reductase (ER) of fungal fatty acid synthase and to 2-nitropropane dioxygenase (2-ND) from Pseudomonas aeruginosa, although there were some differences among these structures. We determined the crystal structure of FabK in complex with a phenylimidazole derivative inhibitor to envision the binding site interactions. The crystal structure reveals that the inhibitor binds to a hydrophobic pocket in the active site of FabK, and this is accompanied by induced-fit movements of two loop regions. The thiazole ring and part of the ureido moiety of the inhibitor are involved in a face-to-face pi-pi stacking interaction with the isoalloxazine ring of FMN. The side-chain conformation of the proposed catalytic residue, His144, changes upon complex formation. Lineweaver-Burk plots indicate that the inhibitor binds competitively with respect to NADH, and uncompetitively with respect to crotonoyl coenzyme A. We propose that the primary basis of the inhibitory activity is competition with NADH for binding to FabK, which is the first step of the two-step ping-pong catalytic mechanism. PMID:18305197

  19. Analysis of TcdB Proteins within the Hypervirulent Clade 2 Reveals an Impact of RhoA Glucosylation on Clostridium difficile Proinflammatory Activities

    Science.gov (United States)

    Quesada-Gómez, Carlos; López-Ureña, Diana; Chumbler, Nicole; Kroh, Heather K.; Castro-Peña, Carolina; Rodríguez, César; Orozco-Aguilar, Josué; González-Camacho, Sara; Rucavado, Alexandra; Guzmán-Verri, Caterina; Lawley, Trevor D.; Lacy, D. Borden

    2016-01-01

    Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1V) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdBNAP1 and TcdBNAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdBNAP1 induced a stronger proinflammatory response than TcdBNAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains. PMID:26755157

  20. Salt bridges overlapping the gonadotropin-releasing hormone receptor agonist binding site reveal a coincidence detector for G protein-coupled receptor activation.

    Science.gov (United States)

    Janovick, Jo Ann; Pogozheva, Irina D; Mosberg, Henry I; Conn, P Michael

    2011-08-01

    G protein-coupled receptors (GPCRs) play central roles in most physiological functions, and mutations in them cause heritable diseases. Whereas crystal structures provide details about the structure of GPCRs, there is little information that identifies structural features that permit receptors to pass the cellular quality control system or are involved in transition from the ground state to the ligand-activated state. The gonadotropin-releasing hormone receptor (GnRHR), because of its small size among GPCRs, is amenable to molecular biological approaches and to computer modeling. These techniques and interspecies comparisons are used to identify structural features that are important for both intracellular trafficking and GnRHR activation yet distinguish between these processes. Our model features two salt (Arg(38)-Asp(98) and Glu(90)-Lys(121)) and two disulfide (Cys(14)-Cys(200) and Cys(114)-Cys(196)) bridges, all of which are required for the human GnRHR to traffic to the plasma membrane. This study reveals that both constitutive and ligand-induced activation are associated with a "coincidence detector" that occurs when an agonist binds. The observed constitutive activation of receptors lacking Glu(90)-Lys(121), but not Arg(38)-Asp(98) ionic bridge, suggests that the role of the former connection is holding the receptor in the inactive conformation. Both the aromatic ring and hydroxyl group of Tyr(284) and the hydrogen bonding of Ser(217) are important for efficient receptor activation. Our modeling results, supported by the observed influence of Lys(191) from extracellular loop 2 (EL2) and a four-residue motif surrounding this loop on ligand binding and receptor activation, suggest that the positioning of EL2 within the seven-α-helical bundle regulates receptor stability, proper trafficking, and function. PMID:21527534

  1. Analysis of TcdB Proteins within the Hypervirulent Clade 2 Reveals an Impact of RhoA Glucosylation on Clostridium difficile Proinflammatory Activities.

    Science.gov (United States)

    Quesada-Gómez, Carlos; López-Ureña, Diana; Chumbler, Nicole; Kroh, Heather K; Castro-Peña, Carolina; Rodríguez, César; Orozco-Aguilar, Josué; González-Camacho, Sara; Rucavado, Alexandra; Guzmán-Verri, Caterina; Lawley, Trevor D; Lacy, D Borden; Chaves-Olarte, Esteban

    2016-01-01

    Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1V) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdBNAP1 and TcdBNAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdBNAP1 induced a stronger proinflammatory response than TcdBNAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains. PMID:26755157

  2. Studies on Variation of Carotenoid-Proteins Content in Cassava (Manihot esculenta Crantz) Storage Root Reveal Implications for Breeding and the Use of Induced Mutations

    International Nuclear Information System (INIS)

    Carotenoid-Protein content in cassava storage root (CSR) is low but variable, and characterization of this variability is lacking. Accumulation of carotenoids occurs in chromoplast and depends on a broad class of proteins named carotenoid associated proteins (CAP), lipids and the biosynthesis of carotenoids. Twenty-nine landraces and progeny of 200 individuals were accessed for CAP and carotenoid content varied in two ways. First, related to landrace diversity, total buffer extractable proteins (TBEP), buffer insoluble proteins (BIP) and total carotenoid and β-carotene content were assessed. Significant differences were observed in the tested genotypes. Secondly, analyses related to storage root tissue age were assessed by TBEP. This showed protein content decreased and total carotenoid content increased as secondary growth proceeds. Further carotenoid-proteins complex (CPC) identified in carotenoid contrasting landraces showed different proteins profile in SDS-PAGE with proteins size of 18 and 33 kDa in low carotenoid (IAC12.829) and 18-20-30-33 kDa in a high total carotenoid landrace (Cas74.1). Progeny analysis for TBEP and total carotenoid content confirmed the interdependence of carotenoid-proteins association by correlation analysis, estimated heritability of individual traits and grouping clones for carotenoid-proteins content. Results allow us to conclude that: natural carotenoid-protein content varies due to differential genetic background and storage root tissue age; carotenoid-protein complex showed variation in protein and carotenoid types; estimated heritability of proteins and carotenoids traits showed different values. The establishment of a genetic component allows future strategies including traditional breeding and the use of induced mutations to create novel variation for the nutritional improvement of cassava tubers. (author)

  3. Histopathologic characterization of the BTBR mouse model of autistic-like behavior reveals selective changes in neurodevelopmental proteins and adult hippocampal neurogenesis

    Directory of Open Access Journals (Sweden)

    Stephenson Diane T

    2011-05-01

    doublecortin, PSA-NCAM and NeuroD immunoreactive cells in the subgranular zone of the dentate gyrus, and a marked reduction in the number of 5-bromo-2'-deoxyuridine (BrdU positive progenitors. Furthermore, a significant and profound reduction in BDNF mRNA was seen in the BTBR dentate gyrus. No significant differences were seen in the expression of AchE, mossy fiber synapses or immunoreactivities of microtubule-associated protein MAP2, parvalbumin and glutamate decarboxylase GAD65 or GAD67 isoforms. Conclusions We documented modest and selective alterations in glia, neurons and synapses in BTBR forebrain, along with reduced neurogenesis in the adult hippocampus. Of all markers examined, the most distinctive changes were seen in the neurodevelopmental proteins NG2, PSA-NCAM, NeuroD and DCX. Our results are consistent with aberrant development of the nervous system in BTBR mice, and may reveal novel substrates to link callosal abnormalities and autistic behaviors. The changes that we observed in the BTBR mice suggest potential novel therapeutic strategies for intervention in autism spectrum disorders.

  4. Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components

    OpenAIRE

    Ying ZHOU; Asahara, Haruichi; Gaucher, Eric A.; Chong, Shaorong

    2012-01-01

    Thermus thermophilus is a thermophilic model organism distantly related to the mesophilic model organism E. coli. We reconstituted protein translation of Thermus thermophilus in vitro from purified ribosomes, transfer ribonucleic acids (tRNAs) and 33 recombinant proteins. This reconstituted system was fully functional, capable of translating natural messenger RNA (mRNA) into active full-length proteins at temperatures up to 65°C and with yields up to 60 μg/ml. Surprisingly, the synthesis of a...

  5. Molecular Modeling of Lectin-Like Protein from Acacia farnesiana Reveals a Possible Anti-Inflammatory Mechanism in Carrageenan-Induced Inflammation

    OpenAIRE

    Vanessa Erika Ferreira Abrantes; Bruno Anderson Matias da Rocha; Raphael Batista da Nóbrega; José Caetano Silva-Filho; Claudener Souza Teixeira; Benildo Sousa Cavada; Carlos Alberto de Almeida Gadelha; Sergio Henrique Ferreira; Jozi Godoy Figueiredo; Tatiane Santi-Gadelha; Plinio Delatorre

    2013-01-01

    Acacia farnesiana lectin-like protein (AFAL) is a chitin-binding protein and has been classified as phytohaemagglutinin from Phaseolus vulgaris (PHA). Legume lectins are examples for structural studies, and this family of proteins shows a remarkable conservation in primary, secondary, and tertiary structures. Lectins have ability to reduce the effects of inflammation caused by phlogistic agents, such as carrageenan (CGN). This paper explains the anti-inflammatory activity of AFAL through stru...

  6. Cooperative protein structural dynamics of homodimeric hemoglobin linked to water cluster at subunit interface revealed by time-resolved X-ray solution scattering

    Directory of Open Access Journals (Sweden)

    Jong Goo Kim

    2016-03-01

    Full Text Available Homodimeric hemoglobin (HbI consisting of two subunits is a good model system for investigating the allosteric structural transition as it exhibits cooperativity in ligand binding. In this work, as an effort to extend our previous study on wild-type and F97Y mutant HbI, we investigate structural dynamics of a mutant HbI in solution to examine the role of well-organized interfacial water cluster, which has been known to mediate intersubunit communication in HbI. In the T72V mutant of HbI, the interfacial water cluster in the T state is perturbed due to the lack of Thr72, resulting in two less interfacial water molecules than in wild-type HbI. By performing picosecond time-resolved X-ray solution scattering experiment and kinetic analysis on the T72V mutant, we identify three structurally distinct intermediates (I1, I2, and I3 and show that the kinetics of the T72V mutant are well described by the same kinetic model used for wild-type and F97Y HbI, which involves biphasic kinetics, geminate recombination, and bimolecular CO recombination. The optimized kinetic model shows that the R-T transition and bimolecular CO recombination are faster in the T72V mutant than in the wild type. From structural analysis using species-associated difference scattering curves for the intermediates, we find that the T-like deoxy I3 intermediate in solution has a different structure from deoxy HbI in crystal. In addition, we extract detailed structural parameters of the intermediates such as E-F distance, intersubunit rotation angle, and heme-heme distance. By comparing the structures of protein intermediates in wild-type HbI and the T72V mutant, we reveal how the perturbation in the interfacial water cluster affects the kinetics and structures of reaction intermediates of HbI.

  7. Proteomics analysis in mature seed of four peanut cultivars using two-dimensional gel electrophoresis reveals distinct differential expression of storage, anti-nutritive, and allergenic proteins

    Science.gov (United States)

    Protein profiles of total seed proteins isolated from mature seeds of four peanut cultivars, New Mexico Valencia C (NM Valencia C), Tamspan 90, Georgia Green, and NC-7, were studied using two-dimensional gel electrophoresis coupled with nano electrospray ionization liquid chromatography tandem mass ...

  8. Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed.

    Science.gov (United States)

    Dale, Renee; Ohmuro-Matsuyama, Yuki; Ueda, Hiroshi; Kato, Naohiro

    2016-01-01

    The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively. PMID:26886551

  9. Studies on variation of carotenoid-proteins content in Cassava (Manihot esculenta Crantz) storage root reveal implications for breeding and the use of induced mutations

    Science.gov (United States)

    Protein content in storage roots of cassava is low but variable and characterization of this variability is lacking. Total buffer extractable proteins (TBEP) content in pigmented cassava landraces varied from 0.9-7.5 (mg/gDWt.) and correlated with total carotenoid content (R2=0.4757). More than 3x T...

  10. Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Shadab Anwar

    Full Text Available Iron-Sulfur (Fe-S proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1 protein and Nucleotide binding protein 35 (Nbp35. In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151 of Nbp35 and (G5-V6, M34-D39 and G46-A52 of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins

  11. Proteomics analysis reveals distinct involvement of embryo and endosperm proteins during seed germination in dormant and non-dormant rice seeds.

    Science.gov (United States)

    Xu, Heng-Heng; Liu, Shu-Jun; Song, Shun-Hua; Wang, Rui-Xia; Wang, Wei-Qing; Song, Song-Quan

    2016-06-01

    Seed germination is a complex trait which is influenced by many genetic, endogenous and environmental factors, but the key event(s) associated with seed germination are still poorly understood. In present study, the non-dormant cultivated rice Yannong S and the dormant Dongxiang wild rice seeds were used as experimental materials, we comparatively investigated the water uptake, germination time course, and the differential proteome of the effect of embryo and endosperm on germination of these two types of seeds. A total of 231 and 180 protein spots in embryo and endosperm, respectively, showed a significant change in abundance during germination. We observed that the important proteins associated with seed germination included those involved in metabolism, energy production, protein synthesis and destination, storage protein, cell growth and division, signal transduction, cell defense and rescue. The contribution of embryo and endosperm to seed germination is different. In embryo, the proteins involved in amino acid activation, sucrose cleavage, glycolysis, fermentation and protein synthesis increased; in endosperm, the proteins involved in sucrose cleavage and glycolysis decreased, and those with ATP and CoQ synthesis and proteolysis increased. Our results provide some new knowledge to understand further the mechanism of seed germination. PMID:27035683

  12. High-resolution two-dimensional polyacrylamide gel electrophoresis reveals a glucose-response protein of 65 kDa in pancreatic islet cells

    International Nuclear Information System (INIS)

    High-resolution two-dimensional PAGE was used to search for glucose-response proteins in isolated pancreatic islets that were labeled with [35S]methionine at ambient glucose concentrations of 0-18 mM. A 65-kDa protein, isoelectric focusing point of approximately 6.6-7.0, was discovered that showed at least a 20-fold stimulation of radiolabeling when glucose in the labeling medium was increased from 3 to 18 mM, in contrast to a 2.5-fold enhancement of label incorporation into total islet proteins. This 65-kDa protein is evident after 30 min of labeling with 18 mM glucose and is preferentially synthesized compared to its nearest neighbors after both 30 and 60 min of labeling. Glucose induction of the 65-kDa protein was virtually blocked by D-mannoheptulose. Glucose induction of this 65-kDa protein is in practically all aspects comparable to glucose induction of insulin and glucokinase in pancreatic beta cells. A working hypothesis is developed proposing that glucose-response proteins or glucospondins are pivotal constituents of pancreatic islet cells and that their discovery and exploration promise new insights into normal and pathological islet cell function

  13. Salinity Stress in Roots of Contrasting BarleyGenotypes Reveals Time-Distinct and Genotype-Specific Patterns for Defined Proteins

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    Soil salinity is one of the most severe abiotic stress factors threatening agriculture worldwide. Hence,particular interest exists in unraveling mechanisms leading to salt tolerance and improved crop plant performance onsaline soils. Barley is considered to be one of the most salinity-tolerant crops, but varying levels of tolerance are wellcharacterized. A proteomic analysis of the roots of two contrasting cultivars (cv. Steptoe and cv. Morex) is presented.Young plants were exposed to a period of 1, 4, 7, or 10 d at 0, 100, or 150mM NaCI. The root proteome was analyzedbased on two-dimensional gel electrophoresis. A number of cultivar-specific and salinity stress-responsive proteins wereidentified. Mass spectrometry-based identification was successful for 74 proteins, and a hierarchical clustering analysisgrouped these into five clusters based on similarity of expression profile. The rank product method was applied to sta-tistically access the early and late responses, and this delivered a number of new candidate proteins underlying salinitytolerance in barley. Among these were some germin-like proteins, some pathogenesis-related proteins, and numerousas-yet uncharacterized proteins. Notably, proteins involved in detoxification pathways and terpenoid biosynthesis weredetected as early responsive to salinity and may function as a means of modulating growth-regulating mechanisms andmembrane stability via fine tuning of phytohormone and secondary metabolism in the root.

  14. Comparative proteomics of chloroplasts envelopes from bundle sheath and mesophyll chloroplasts reveals novel membrane proteins with a possible role in C4-related metabolite fluxes and development.

    Directory of Open Access Journals (Sweden)

    Kalpana eManandhar-Shrestha

    2013-03-01

    Full Text Available As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second green revolution will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M and bundle sheath (BS chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts (cp. 74% have a known or predicted membrane association. 21 membrane proteins were 2-15 times more abundant in BS cells, while 36 proteins were more abundant in M cp envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of thirteen candidate genes. Cp association was confirmed using GFP labeling. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene expression is increased in the immature part of the leaf and was lower in the fully developed parts of the leaf, suggesting a need for/incorporation of the protein during chloroplast

  15. Proteomics Reveals that Proteins Expressed During the Early Stage of Bacillus anthracis Infection Are Potential Targets for the Development of Vaccines and Drugs

    Institute of Scientific and Technical Information of China (English)

    Chun-Ming Huang; Craig A. Elmets; De-chu C. Tang; Fuming Li; Nabiha Yusuf

    2004-01-01

    In this review, we advance a new concept in developing vaccines and/or drugs to target specific proteins expressed during the early stage of Bacillus anthracis (an thrax) infection and address existing challenges to this concept. Three proteins (immune inhibitor A, GPR-like spore protease, and alanine racemase) initially identified by proteomics in our laboratory were found to have differential expres sions during anthrax spore germination and early outgrowth. Other studies of different bacillus strains indicate that these three proteins are involved in either germination or cytotoxicity of spores, suggesting that they may serve as potential targets for the design of anti-anthrax vaccines and drugs.

  16. Identification of a TAL1 Target Gene Reveals a Positive Role for the LIM Domain-Binding Protein Ldb1 in Erythroid Gene Expression and Differentiation

    OpenAIRE

    Xu, Zhixiong; Huang, Suming; Chang, Long-Sheng; Agulnick, Alan D.; Brandt, Stephen J.

    2003-01-01

    The TAL1 (or SCL) gene, originally identified from its involvement by a recurrent chromosomal translocation, encodes a basic helix-loop-helix transcription factor essential for erythropoiesis. Although presumed to regulate transcription, its target genes are largely unknown. We show here that a nuclear complex containing TAL1, its DNA-binding partner E47, zinc finger transcription factor GATA-1, LIM domain protein LMO2, and LIM domain-binding protein Ldb1 transactivates the protein 4.2 (P4.2)...

  17. Comprehensive peptidomic and glycomic evaluation reveals that sweet whey permeate from colostrum is a source of milk protein-derived peptides and oligosaccharides

    OpenAIRE

    Dallas, David C.; Weinborn, Valerie; de Moura Bell, Juliana M.L.N.; Wang, Meng; Parker, Evan A.; Guerrero, Andres; Hettinga, Kasper A.; Lebrilla, Carlito B.; German, J. Bruce; Barile, Daniela

    2014-01-01

    Whey permeate is a co-product obtained when cheese whey is passed through an ultrafiltration membrane to concentrate whey proteins. Whey proteins are retained by the membrane, whereas the low-molecular weight compounds such as lactose, salts, oligosaccharides and peptides pass through the membrane yielding whey permeate. Research shows that bovine milk from healthy cows contains hundreds of naturally occurring peptides – many of which are homologous with known antimicrobial and immunomodulato...

  18. Disease-causing Mutation in PKR2 Receptor Reveals a Critical Role of Positive Charges in the Second Intracellular Loop for G-protein Coupling and Receptor Trafficking*

    OpenAIRE

    Peng, Zhen; Tang, Yong; Luo, Hunjin; Jiang, Fang; Yang, Jiannan; Sun, Lin; Li, Jia-Da

    2011-01-01

    Prokineticins are a pair of signal factors involved in many physiological processes by binding to two closely related G-protein-coupled receptors, PKR1 and PKR2. Recently, mutations in prokineticin 2 (PK2) and PKR2 are found to be associated with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, disorders characterized by delayed puberty and infertility. However, little is known how PKRs interact and activate G-proteins to elicit signal transduction. In the present study, we ...

  19. Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans

    OpenAIRE

    De Castro, Cristina; Molinaro, Antonio; Piacente, Francesco; Gurnon, James R.; Sturiale, Luisa; Palmigiano, Angelo; Lanzetta, Rosa; Parrilli, Michelangelo; Garozzo, Domenico; Tonetti, Michela G.; Van Etten, James L.

    2013-01-01

    The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) ...

  20. Label-free LC-MSe in tissue and serum reveals protein networks underlying differences between benign and malignant serous ovarian tumors.

    Directory of Open Access Journals (Sweden)

    Wouter Wegdam

    Full Text Available PURPOSE: To identify proteins and (molecular/biological pathways associated with differences between benign and malignant epithelial ovarian tumors. EXPERIMENTAL PROCEDURES: Serum of six patients with a serous adenocarcinoma of the ovary was collected before treatment, with a control group consisting of six matched patients with a serous cystadenoma. In addition to the serum, homogeneous regions of cells exhibiting uniform histology were isolated from benign and cancerous tissue by laser microdissection. We subsequently employed label-free liquid chromatography tandem mass spectrometry (LC-MSe to identify proteins in these serum and tissues samples. Analyses of differential expression between samples were performed using Bioconductor packages and in-house scripts in the statistical software package R. Hierarchical clustering and pathway enrichment analyses were performed, as well as network enrichment and interactome analysis using MetaCore. RESULTS: In total, we identified 20 and 71 proteins that were significantly differentially expressed between benign and malignant serum and tissue samples, respectively. The differentially expressed protein sets in serum and tissue largely differed with only 2 proteins in common. MetaCore network analysis, however inferred GCR-alpha and Sp1 as common transcriptional regulators. Interactome analysis highlighted 14-3-3 zeta/delta, 14-3-3 beta/alpha, Alpha-actinin 4, HSP60, and PCBP1 as critical proteins in the tumor proteome signature based on their relative overconnectivity. The data have been deposited to the ProteomeXchange with identifier PXD001084. DISCUSSION: Our analysis identified proteins with both novel and previously known associations to ovarian cancer biology. Despite the small overlap between differentially expressed protein sets in serum and tissue, APOA1 and Serotransferrin were significantly lower expressed in both serum and cancer tissue samples, suggesting a tissue-derived effect in serum

  1. Cloning of the cDNA for the TATA-binding protein-associated factorII170 subunit of transcription factor B-TFIID reveals homology to global transcription regulators in yeast and Drosophila

    OpenAIRE

    van der Knaap, Jan A.; Borst, Jan Willem; van der Vliet, Peter C.; Gentz, Reiner; Timmers, H.Th. Marc

    1997-01-01

    The human transcription factor B-TFIID is comprised of TATA-binding protein (TBP) in complex with one TBP-associated factor (TAF) of 170 kDa. We report the isolation of the cDNA for TAFII170. By cofractionation and coprecipitation experiments, we show that the protein encoded by the cDNA encodes the TAF subunit of B-TFIID. Recombinant TAFII170 has (d)ATPase activity. Inspection of its primary structure reveals a striking homology with genes of other organisms, yeast MOT1, and Drosophila moira...

  2. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity

    Directory of Open Access Journals (Sweden)

    Zulezwan A. Malik

    2013-12-01

    Full Text Available Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC coupled to mass spectrometry (MS affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group bred as either high- or low-capacity runners (HCR and LCR, respectively that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001 in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897 and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5. Sixteen proteins were significantly (p < 0.05 more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH was 1

  3. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization.

    Science.gov (United States)

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-01

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement. PMID:26791570

  4. Mechanistic and Structural Studies of Protein-Only RNase P Compared to Ribonucleoproteins Reveal the Two Faces of the Same Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Cédric Schelcher

    2016-06-01

    Full Text Available RNase P, the essential activity that performs the 5′ maturation of tRNA precursors, can be achieved either by ribonucleoproteins containing a ribozyme present in the three domains of life or by protein-only enzymes called protein-only RNase P (PRORP that occur in eukaryote nuclei and organelles. A fast growing list of studies has investigated three-dimensional structures and mode of action of PRORP proteins. Results suggest that similar to ribozymes, PRORP proteins have two main domains. A clear functional analogy can be drawn between the specificity domain of the RNase P ribozyme and PRORP pentatricopeptide repeat domain, and between the ribozyme catalytic domain and PRORP N4BP1, YacP-like Nuclease domain. Moreover, both types of enzymes appear to dock with the acceptor arm of tRNA precursors and make specific contacts with the corner of pre-tRNAs. While some clear differences can still be delineated between PRORP and ribonucleoprotein (RNP RNase P, the two types of enzymes seem to use, fundamentally, the same catalytic mechanism involving two metal ions. The occurrence of PRORP and RNP RNase P represents a remarkable example of convergent evolution. It might be the unique witness of an ongoing replacement of catalytic RNAs by proteins for enzymatic activities.

  5. Plasma Proteins Modified by Advanced Glycation End Products (AGEs) Reveal Site-specific Susceptibilities to Glycemic Control in Patients with Type 2 Diabetes.

    Science.gov (United States)

    Greifenhagen, Uta; Frolov, Andrej; Blüher, Matthias; Hoffmann, Ralf

    2016-04-29

    Protein glycation refers to the reversible reaction between aldoses (or ketoses) and amino groups yielding relatively stable Amadori (or Heyns) products. Consecutive oxidative cleavage reactions of these products or the reaction of amino groups with other reactive substances (e.g. α-dicarbonyls) yield advanced glycation end products (AGEs) that can alter the structures and functions of proteins. AGEs have been identified in all organisms, and their contents appear to rise with some diseases, such as diabetes and obesity. Here, we report a pilot study using highly sensitive and specific proteomics approach to identify and quantify AGE modification sites in plasma proteins by reversed phase HPLC mass spectrometry in tryptic plasma digests. In total, 19 AGE modification sites corresponding to 11 proteins were identified in patients with type 2 diabetes mellitus under poor glycemic control. The modification degrees of 15 modification sites did not differ among cohorts of normoglycemic lean or obese and type 2 diabetes mellitus patients under good and poor glycemic control. The contents of two amide-AGEs in human serum albumin and apolipoprotein A-II were significantly higher in patients with poor glycemic control, although the plasma levels of both proteins were similar among all plasma samples. These two modification sites might be useful to predict long term, AGE-related complications in diabetic patients, such as impaired vision, increased arterial stiffness, or decreased kidney function. PMID:26933035

  6. Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize.

    Science.gov (United States)

    Wu, Xiaolin; Gong, Fangping; Yang, Le; Hu, Xiuli; Tai, Fuju; Wang, Wei

    2014-01-01

    ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5), deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs), late embryogenesis abundant (LEA) proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation. PMID:25653661

  7. Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize

    Directory of Open Access Journals (Sweden)

    Xiaolin eWu

    2015-01-01

    Full Text Available ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5, deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs, late embryogenesis abundant (LEA proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation.

  8. What a Difference Melphalan Makes! Differential Protein Expression in Melphalan Sensitive and Resistant Multiple Myeloma Cells Revealed by SILAC-based Quantitative Proteomics

    DEFF Research Database (Denmark)

    Zub, Kamila Anna; Sousa, Mirta M.L.; Young, Clifford;

    have been suggested to contribute to drug resistance, including modulated drug transport and metabolism, enhanced DNA repair and decreased apoptotic signalling1,2,3. Major common determinants associated with melphalan resistance, however, remain elusive. To further identify potential protein biomarkers...... and/or pathways involved in elevated drug tolerance, SILAC-based quantitative protein profiling was performed using the multiple myeloma cell line RPMI 8226 (sensitive) and its derivative RPMI 8226-LR5 (adapted to growth in melphalan). By this approach, 2827 proteins were identified and 1163 were......One of the most frequently used and effective drugs against multiple myeloma is the alkylating drug melphalan. However, the treatment is often hampered by development of drug resistance. Eventually, nearly all patients develop resistance, and mean survival time is about 3 years. Several factors...

  9. The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4

    International Nuclear Information System (INIS)

    Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting β-hairpin observed in other polyomaviruses. We postulate that the terminal β-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.

  10. Directed Evolution Reveals the Binding Motif Preference of the LC8/DYNLL Hub Protein and Predicts Large Numbers of Novel Binders in the Human Proteome

    OpenAIRE

    Rapali, Péter; Radnai, László; Süveges, Dániel; Harmat, Veronika; Tölgyesi, Ferenc; Weixiao Y. Wahlgren; Katona, Gergely; Nyitray, László; Pál, Gábor

    2011-01-01

    LC8 dynein light chain (DYNLL) is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [D/S]-4K-3X-2[T/V/I]-1Q0[T/V]1[D/E]2. The motifs are localized in disordered segments of the DYNLL-binding proteins and are often flanked by coiled...

  11. Structural and functional studies of the biotin protein ligase from Aquifex aeolicus reveal a critical role for a conserved residue in target specificity

    OpenAIRE

    Tron, Cecile M; McNae, Iain W.; Nutley, Margaret; Clarke, David J; Cooper, Alan; Walkinshaw, Malcolm D.; Baxter, Robert L.; Campopiano, Dominic J.

    2009-01-01

    Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5'-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 A resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed β- and γ-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal dom...

  12. Zebrafish Models for the Mechanosensory Hair Cell Dysfunction in Usher Syndrome 3 Reveal That Clarin-1 Is an Essential Hair Bundle Protein

    OpenAIRE

    Gopal, Suhasini R.; Chen, Daniel H.-C.; Chou, Shih-Wei; Zang, Jingjing; Neuhauss, Stephan C. F.; Stepanyan, Ruben; McDermott, Brian M., Jr.; Alagramam, Kumar N.

    2015-01-01

    Usher syndrome type III (USH3) is characterized by progressive loss of hearing and vision, and varying degrees of vestibular dysfunction. It is caused by mutations that affect the human clarin-1 protein (hCLRN1), a member of the tetraspanin protein family. The missense mutation CLRN1N48K, which affects a conserved N-glycosylation site in hCLRN1, is a common causative USH3 mutation among Ashkenazi Jews. The affected individuals hear at birth but lose that function over time. Here, we developed...

  13. Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs

    OpenAIRE

    Geerds, Christina; Wohlmann, Jens; Haas, Albert; Niemann, Hartmut H.

    2014-01-01

    Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD ph...

  14. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

    Science.gov (United States)

    Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

    2013-12-01

    Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly (p ion (551.21 m/z) of the doubly-charged peptide SLGVGFATR (454.19 m/z) of residues 23-31 of FABPH. SRM was conducted on technical replicates of each biological sample and exhibited a coefficient of variation of 20%. The abundance of FABPH measured by SRM was 2.84-fold greater (p = 0.0095) in HCR muscle. In addition, SRM of FABPH was performed in vastus lateralis samples of young and elderly humans with different habitual activity levels (collected during a previous study) finding FABPH abundance was 2.23-fold greater (p = 0.0396) in endurance-trained individuals regardless of differences in age. In summary, our findings in HCR/LCR rats provide protein-level confirmation for earlier

  15. Expression of human Cfdp1 gene in Drosophila reveals new insights into the function of the evolutionarily conserved BCNT protein family

    Science.gov (United States)

    Messina, Giovanni; Atterrato, Maria Teresa; Fanti, Laura; Giordano, Ennio; Dimitri, Patrizio

    2016-01-01

    The Bucentaur (BCNT) protein family is widely distributed in eukaryotes and is characterized by a highly conserved C-terminal domain. This family was identified two decades ago in ruminants, but its role(s) remained largely unknown. Investigating cellular functions and mechanism of action of BCNT proteins is challenging, because they have been implicated in human craniofacial development. Recently, we found that YETI, the D. melanogaster BCNT, is a chromatin factor that participates to H2A.V deposition. Here we report the effects of in vivo expression of CFDP1, the human BCNT protein, in Drosophila melanogaster. We show that CFDP1, similarly to YETI, binds to chromatin and its expression results in a wide range of abnormalities highly reminiscent of those observed in Yeti null mutants. This indicates that CFDP1 expressed in flies behaves in a dominant negative fashion disrupting the YETI function. Moreover, GST pull-down provides evidence indicating that 1) both YETI and CFDP1 undergo homodimerization and 2) YETI and CFDP1 physically interact each other by forming inactive heterodimers that would trigger the observed dominant-negative effect. Overall, our findings highlight unanticipated evidences suggesting that homodimerization mediated by the BCNT domain is integral to the chromatin functions of BCNT proteins. PMID:27151176

  16. Neutron scattering with deuterium labeling reveals the nature of complexes formed by Ca{sup 2+}-binding proteins and their regulatory targets

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J. [Los Alamos National Laboratory, NM (United States)

    1994-12-01

    Small-angle neutron scattering with deuterium labeling is extremely useful for studying the structures of complex biomolecular assemblies in solution. The different neutron scattering properties of their isotopes of hydrogen combines with the ability to uniformly label biomolecules with deuterium allow one to characterize the structures and relative dispositions of the individual components of an assembly using methods of {open_quotes}contrast variation.{close_quotes} We have applied these techniques to studies of the evolutionarily related dumbbell-shaped Ca{sup 2+}-binding proteins calmodulin and troponin C and their interactions with the target proteins whose activities they regulate. Ca{sup 2+} is one of the simplest of nature`s messengers used in the communication pathways between physiological stimulus and cellular response. The signaling mechanism generally involves Ca{sup 2+} binding to a protein and inducing a conformational change that transmits a signal to modify the activity of a specific target protein. Ca{sup 2+} is thus important in the regulation of a diverse array of intracellular responses, including neurotransmitter release, muscle contraction, the degradation of glycogen to glucose to generate energy, microtubule assembly, membrane phosphorylation, etc. It is the conformational language of the Ca{sup 2+} induced signal transduction that we have sought to understand because of its central importance to biochemical regulation and, hence, to healthy cellular function.

  17. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    International Nuclear Information System (INIS)

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer

  18. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Parent, Kristin N., E-mail: kparent@msu.edu [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Tang, Jinghua; Cardone, Giovanni [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Gilcrease, Eddie B. [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Janssen, Mandy E.; Olson, Norman H. [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Casjens, Sherwood R., E-mail: sherwood.casjens@path.utah.edu [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Baker, Timothy S., E-mail: tsb@ucsd.edu [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); University of California, San Diego, Division of Biological Sciences, La Jolla, CA, 92093 (United States)

    2014-09-15

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer.

  19. HydPred: a novel method for the identification of protein hydroxylation sites that reveals new insights into human inherited disease.

    Science.gov (United States)

    Li, Shuyan; Lu, Jun; Li, Jiazhong; Chen, Ximing; Yao, Xiaojun; Xi, Lili

    2016-01-26

    The disruption of protein hydroxylation is highly associated with several serious diseases and consequently the identification of protein hydroxylation sites has attracted significant attention recently. Here, we report the development of an improved method, called HydPred, to identify protein hydroxylation sites (hydroxyproline and hydroxylysine) based on the synthetic minority over-sampling technique (SMOTE), the random forest (RF) algorithm and four blocks of newly composed features that are derived from the protein primary sequence. The HydPred method achieved the best prediction performance reported until now with Matthew's correlation coefficient values of 0.770 and 0.857 for hydroxyproline and hydroxylysine, respectively, according to jack-knife cross-validation. This represents an improvement of 8% for hydroxyproline and 19% for hydroxylysine compared to the best results of available predictors. The prediction performance of HydPred for the external validation of hydroxyproline and hydroxylysine was also improved compared with other published methods. We subsequently applied HydPred to study the association of disruption of hydroxylation sites with human inherited disease. The analyses suggested that the loss of hydroxylation sites is more likely to cause disease instead of the gain of hydroxylation sites and 52 different human inherited diseases were found to be highly associated with the loss of hydroxylation sites. Therefore, HydPred represents a new strategy to discover the molecular basis of pathogenesis associated with abnormal hydroxylation. HydPred is now available online as a user-friendly web server at . PMID:26661679

  20. A Click Chemistry‐Based Proteomic Approach Reveals that 1,2,4‐Trioxolane and Artemisinin Antimalarials Share a Common Protein Alkylation Profile

    Science.gov (United States)

    Ismail, Hanafy M.; Barton, Victoria E.; Panchana, Matthew; Charoensutthivarakul, Sitthivut; Biagini, Giancarlo A.; Ward, Stephen A.

    2016-01-01

    Abstract In spite of the recent increase in endoperoxide antimalarials under development, it remains unclear if all these chemotypes share a common mechanism of action. This is important since it will influence cross‐resistance risks between the different classes. Here we investigate this proposition using novel clickable 1,2,4‐trioxolane activity based protein‐profiling probes (ABPPs). ABPPs with potent antimalarial activity were able to alkylate protein target(s) within the asexual erythrocytic stage of Plasmodium falciparum (3D7). Importantly, comparison of the alkylation fingerprint with that generated from an artemisinin ABPP equivalent confirms a highly conserved alkylation profile, with both endoperoxide classes targeting proteins in the glycolytic, hemoglobin degradation, antioxidant defence, protein synthesis and protein stress pathways, essential biological processes for plasmodial survival. The alkylation signatures of the two chemotypes show significant overlap (ca. 90 %) both qualitatively and semi‐quantitatively, suggesting a common mechanism of action that raises concerns about potential cross‐resistance liabilities. PMID:27397940

  1. Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs.

    Science.gov (United States)

    Geerds, Christina; Wohlmann, Jens; Haas, Albert; Niemann, Hartmut H

    2014-07-01

    Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology. PMID:25005079

  2. Regulation of mammary stem cell population with dietary intake of soy protein isolate reveals novel mechanisms for diet-mediated control of mammary tumorigenesis

    Science.gov (United States)

    Breast cancer risk is highly modified by environmental factors including diet. Previously, we showed that dietary intake of soy protein isolate (SPI) decreased mammary tumor incidence and increased mammary tumor latency in rats relative to those fed a control casein (CAS) diet, when exposed to the c...

  3. Quantitative Proteome Analysis Reveals Increased Content of Basement Membrane Proteins in Arteries from Patients with Type 2 Diabetes and Lower Levels among Metformin Users

    DEFF Research Database (Denmark)

    Rørdam Preil, Simone; Kristensen, Lars P; Beck, Hans C;

    2015-01-01

    BACKGROUND: -The increased risk of cardiovascular diseases (CVD) in type 2 diabetes has been extensively documented, but the origins of the association remain largely unknown. We sought to determine changes in protein expressions in arterial tissue from patients with type 2 diabetes and moreover...

  4. Expression of human Cfdp1 gene in Drosophila reveals new insights into the function of the evolutionarily conserved BCNT protein family.

    Science.gov (United States)

    Messina, Giovanni; Atterrato, Maria Teresa; Fanti, Laura; Giordano, Ennio; Dimitri, Patrizio

    2016-01-01

    The Bucentaur (BCNT) protein family is widely distributed in eukaryotes and is characterized by a highly conserved C-terminal domain. This family was identified two decades ago in ruminants, but its role(s) remained largely unknown. Investigating cellular functions and mechanism of action of BCNT proteins is challenging, because they have been implicated in human craniofacial development. Recently, we found that YETI, the D. melanogaster BCNT, is a chromatin factor that participates to H2A.V deposition. Here we report the effects of in vivo expression of CFDP1, the human BCNT protein, in Drosophila melanogaster. We show that CFDP1, similarly to YETI, binds to chromatin and its expression results in a wide range of abnormalities highly reminiscent of those observed in Yeti null mutants. This indicates that CFDP1 expressed in flies behaves in a dominant negative fashion disrupting the YETI function. Moreover, GST pull-down provides evidence indicating that 1) both YETI and CFDP1 undergo homodimerization and 2) YETI and CFDP1 physically interact each other by forming inactive heterodimers that would trigger the observed dominant-negative effect. Overall, our findings highlight unanticipated evidences suggesting that homodimerization mediated by the BCNT domain is integral to the chromatin functions of BCNT proteins. PMID:27151176

  5. Fate of the insecticidal Cry1Ab protein of GM crops in two agricultural soils as revealed by ¹⁴C-tracer studies.

    Science.gov (United States)

    Valldor, Petra; Miethling-Graff, Rona; Martens, Rainer; Tebbe, Christoph C

    2015-09-01

    Insecticidal delta-endotoxins of Bacillus thuringiensis are among the most abundant recombinant proteins released by genetically modified (GM) crops into agricultural soils worldwide. However, there is still controversy about their degradation and accumulation in soils. In this study, (14)C-labelled Cry1Ab protein was applied to soil microcosms at two concentrations (14 and 50 μg g(-1) soil) to quantify the mineralization of Cry1Ab, its incorporation into the soil microbial biomass, and its persistence in two soils which strongly differed in their texture but not in silt or pH. Furthermore, ELISA was used to quantify Cry1Ab and its potential immunoreactive breakdown products in aqueous soil extracts. In both soils, (14)CO2-production was initially very high and then declined during a total monitoring period of up to 135 days. A total of 16 to 23 % of the (14)C activity was incorporated after 29 to 37 days into the soil microbial biomass, indicating that Cry1Ab protein was utilized by microorganisms as a growth substrate. Adsorption in the clay-rich soil was the most important factor limiting microbial degradation; as indicated by higher degradation rates in the more sandy soil, extremely low concentrations of immunoreactive Cry1Ab molecules in the soils' aqueous extracts and a higher amount of (14)C activity bound to the soil with more clay. Ecological risk assessments of Bt-crops should therefore consider that the very low concentrations of extractable Cry1Ab do not reflect the actual elimination of the protein from soils but that, on the other hand, desorbed proteins mineralize quickly due to efficient microbial degradation. PMID:25967657

  6. Resolution of complex fluorescence spectra of lipids and nicotinic acetylcholine receptor by multivariate analysis reveals protein-mediated effects on the receptor's immediate lipid microenvironment

    CERN Document Server

    Wenz, Jorge J; 10.1186/1757-5036-1-6

    2009-01-01

    Analysis of fluorescent spectra from complex biological systems containing various fluorescent probes with overlapping emission bands is a challenging task. Valuable information can be extracted from the full spectra, however, by using multivariate analysis (MA) of measurements at different wavelengths. We applied MA to spectral data of purified Torpedo nicotinic acetylcholine receptor (AChR) protein reconstituted into liposomes made up of dioleoylphosphatidic acid (DOPA) and dioleoylphosphatidylcholine (DOPC) doped with two extrinsic fluorescent probes (NBD-cholesterol/pyrene-PC). Forster resonance energy transfer (FRET) was observed between the protein and pyrene-PC and between pyrene-PC and NBD-cholesterol, leading to overlapping emission bands. Partial least squares analysis was applied to ...

  7. On the Exciton Coupling between Two Chlorophyll Pigments in the Absence of a Protein Environment: Intrinsic Effects Revealed by Theory and Experiment.

    Science.gov (United States)

    Milne, Bruce F; Kjaer, Christina; Houmøller, Jørgen; Stockett, Mark H; Toker, Yoni; Rubio, Angel; Nielsen, Steen Brøndsted

    2016-05-17

    Exciton coupling between two or more chlorophyll (Chl) pigments is a key mechanism associated with the color tuning of photosynthetic proteins but it is difficult to disentangle this effect from shifts that are due to the protein microenvironment. Herein, we report the formation of the simplest coupled system, the Chl a dimer, tagged with a quaternary ammonium ion by electrospray ionization. Based on action spectroscopic studies in vacuo, the dimer complexes were found to absorb 50-70 meV to the red of the monomers under the same conditions. First-principles calculations predict shifts that somewhat depend on the relative orientation of the two Chl units, namely 50 and 30 meV for structures where the Chl rings are stacked and unstacked, respectively. Our work demonstrates that Chl association alone can produce a large portion of the color shift observed in photosynthetic macromolecular assemblies. PMID:27072666

  8. Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Nawrocki, Arkadiusz; Jensen, Steffen Grann;

    2014-01-01

    Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line...... T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13-16-fold increased exosome yield and facilitated...... quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ- proteomics. We identified several proteins linked...

  9. Proteomics of the oxidative stress response induced by hydrogen peroxide and paraquat reveals a novel AhpC-like protein in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Hare, Nathan J; Scott, Nichollas E; Shin, Eun Hye H;

    2011-01-01

    Pseudomonas aeruginosa is a ubiquitous pathogen most typically associated with wound infections, but also the main cause of mortality in patients suffering from cystic fibrosis (CF). The ability to adapt to oxidative stress associated with host immune defense may be one mechanism by which P....... aeruginosa establishes infection in the cystic fibrosis lung and eventually out-competes other pathogenic bacteria to persist into chronic infection. We utilized a proteomics approach to identify the proteins associated with the oxidative stress response of P. aeruginosa PAO1 to hydrogen peroxide...... by hypothetical protein PA3529 following treatment with 10 mM H(2) O(2) . AhpC belongs to the 2-Cys peroxiredoxin family and is a redox enzyme responsible for removing peroxides in bacterial cells. MS analysis showed that PA3529 was altered by the formation of a dimer via a disulfide bond in a manner analogous...

  10. High-resolution structures of the D-alanyl carrier protein (Dcp) DltC from Bacillus subtilis reveal equivalent conformations of apo- and holo-forms.

    Science.gov (United States)

    Zimmermann, Stephan; Pfennig, Sabrina; Neumann, Piotr; Yonus, Huma; Weininger, Ulrich; Kovermann, Michael; Balbach, Jochen; Stubbs, Milton T

    2015-08-19

    D-Alanylation of lipoteichoic acids plays an important role in modulating the properties of Gram-positive bacteria cell walls. The D-alanyl carrier protein DltC from Bacillus subtilis has been solved in apo- and two cofactor-modified holo-forms, whereby the entire phosphopantetheine moiety is defined in one. The atomic resolution of the apo-structure allows delineation of alternative conformations within the hydrophobic core of the 78 residue four helix bundle. In contrast to previous reports for a peptidyl carrier protein from a non-ribosomal peptide synthetase, no obvious structural differences between apo- and holo-DltC forms are observed. Solution NMR spectroscopy confirms these findings and demonstrates in addition that the two forms exhibit similar backbone dynamics on the ps-ns and ms timescales. PMID:26193422